Methods of processing a fluid including a recombinant therapeutic protein and use thereof

ABSTRACT

Provided herein are methods of processing a fluid that include the use of one or both of a circuit system including a tangential flow filtration (TFF) unit and a circuit system including a tangential flow virus filtration (TFVF) unit. Also provided are integrated and continuous processes for manufacturing a therapeutic protein drug substance that include a step of processing a fluid including the recombinant therapeutic protein using any of the methods provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/375,689, filed Aug. 16, 2016; the entire contents of whichare herein incorporated by reference.

TECHNICAL FIELD

This invention relates to methods of biotechnology and thebiomanufacturing of recombinant proteins.

BACKGROUND

Mammalian cells containing a nucleic acid that encodes a recombinantprotein are often used to produce therapeutically or commerciallyimportant proteins. In the current environment of diverse productpipelines, biotechnology companies are increasingly driven to developinnovative solutions for highly flexible and cost-effectivemanufacturing of therapeutic protein drug substances.

SUMMARY

The present invention is based, at least in part, on the discovery thatcircuit systems including a tangential flow filtration (TFF) unit or atangential flow virus filtration (TFVF) unit can be used to continuouslyperform the unit operation of ultrafiltration/diafiltration and virusfiltration, respectively. In view of this discovery, provided herein aremethods of processing a fluid that include the use of one or both of acircuit system including a tangential flow filtration (TFF) unit and acircuit system including a tangential flow virus filtration (TFVF) unit.Also provided are integrated and continuous processes for manufacturinga therapeutic protein drug substance that include a step of processing afluid including the recombinant therapeutic protein using any of themethods provided herein.

Provided herein are methods of processing a fluid including arecombinant therapeutic protein that include: (a) providing a circuitsystem including (i) a tangential flow filtration (TFF) unit havingfirst and second inlets, and (ii) a conduit in fluid communicationbetween the first and second inlets, including at least one port forflowing fluid into or out of, or both, of the system, where the systemis configured such that fluid can be flowed through the conduit and theTFF unit, and filtrate not including the recombinant therapeutic proteincan be collected from the TFF unit; (b) continuously flowing a fluidincluding a recombinant therapeutic protein into the circuit systemthrough one of the at least one port, and discarding filtrate notincluding the recombinant therapeutic protein for a first period oftime; (c) continuously flowing a diafiltration medium into the circuitsystem through one of the at least one port, and discarding filtrate notincluding the recombinant therapeutic protein for a second period oftime; and (d) collecting a fluid including the recombinant therapeuticprotein that is present in the circuit system after the first and secondtime periods through one of the at least one port. In some embodimentsof these methods, step (b) includes continuously flowing the fluid intothe circuit system at a rate of about 0.1 mL/minute to about 100L/minute (e.g., about 0.5 mL/minute to about 200 mL/minute). In someembodiments of these methods of these methods, the first period of timeis about 0.1 minute to about 12 hours (e.g., about 10 minutes to about 4hours). In some embodiments of these methods, the flowing of the fluidinto the circuit system in step (b) occurs unidirectionally. In someembodiments of these methods, step (c) includes continuously flowing thediafiltration medium into the circuit system at a rate of about 0.1mL/minute to about 100 L/minute (e.g., about 0.5 mL/minute to about 200mL/minute). In some embodiments of these methods, the second period oftime is about 5 minutes to about 12 hours (e.g., about 10 minutes toabout 4 hours). In some embodiments of these methods, the flowing of thediafiltration medium into the circuit system in step (c) occursunidirectionally. In some embodiments of these methods, the conduit hasan inner diameter of about 5 mm to about 5 cm (e.g., about 10 mm toabout 3 cm). In some embodiments of these methods, the TFF unitcomprises one or more tangential filter(s) having a surface area ofabout 0.1 cm² to about 50 cm² (e.g., about 0.5 cm² to about 20 cm²). Insome embodiments of these methods, the circuit system further includes apump (e.g., a peristaltic pump) disposed in the conduit.

Some embodiments of these methods further include: (e) providing asecond circuit system including (i) a tangential flow virus filtration(TFVF) unit having first and second inlets, and (ii) a conduit in fluidcommunication between the first and second inlets of the TFVF unit,including at least one port for flowing fluid into or out of, or both,of the second system, where the second system is configured such thatfluid can be flowed through the conduit and the TFVF unit, and filtrateincluding the recombinant therapeutic protein can be collected from theTFVF unit; and (f) flowing the collected fluid of step (d) into thesecond system using a connecting conduit in fluid communication betweena port in the system and a port in the second system, and collectingfiltrate including the recombinant therapeutic protein from the TFVFunit for a third period of time. In some embodiments of these methods,step (f) includes flowing the fluid into the second system at a rate ofabout 0.1 mL/minute to about 100 L/minute (e.g., about 0.5 mL/minute toabout 200 mL/minute). In some embodiments of these methods, the thirdperiod of time is about 0.1 minute to about 12 hours (e.g., about 10minutes to about 2 hours). In some embodiments of these methods, theflowing of the fluid into the second system in step (f) occursunidirectionally. In some embodiments of these methods, the conduit influid communication between the first and second inlets of the TFVF unithas an inner diameter of about 5 mm to about 5 cm (e.g., about 10 mm toabout 3 cm). In some embodiments of these methods, the TFVF unitincludes one or more tangential virus filter(s) having a surface area ofabout 0.1 cm² to about 100 m² (e.g., about 0.5 cm² to about 200 cm²). Insome embodiments of these methods, the connecting conduit has an innerdiameter of about 5 mm to about 10 cm (e.g., about 10 mm to about 5 cm).In some embodiments of these methods, the second system further includesa pump (e.g., a peristaltic pump) disposed in the conduit in fluidcommunication between the first and second inlets of the TFVF unit.

Also provided are methods for processing a fluid including a recombinanttherapeutic protein that include: (a) providing a circuit systemincluding (i) a tangential flow virus filtration (TFVF) unit havingfirst and second inlets, and (ii) a conduit in fluid communicationbetween the first and second inlets of the TFVF unit, including at leastone port for flowing fluid into or out of, or both, of the system, wherethe system is configured such that fluid can be flowed through theconduit and the TFVF unit, and filtrate including the recombinanttherapeutic protein can be collected from the TFVF unit; (b)continuously flowing a fluid including a recombinant therapeutic proteininto the circuit system through one of the at least one port, andcollecting filtrate including the recombinant therapeutic protein fromthe TFVF unit for a period of time. In some embodiments of thesemethods, step (b) includes continuously flowing the fluid into thesystem at a rate of about 0.1 mL/minute to about 100 L/minute (e.g.,about 0.5 mL/minute to about 200 mL/minute). In some embodiments ofthese methods, the period of time is about 5 minutes to about 12 hours(e.g., about 10 minutes to about 3 hours). In some embodiments of thesemethods, the flowing of the fluid into the system in step (b) occursunidirectionally. In some embodiments of these methods, the conduit hasan inner diameter of about 5 mm to about 5 cm (e.g., about 10 mm toabout 3 cm). In some embodiments of these methods, the TFVF unitincludes one or more tangential virus filter(s) having a surface area ofabout 0.1 cm² to about 100 m² (e.g., about 0.5 cm² to about 200 cm²). Insome embodiments of these methods, the system further includes a pump(e.g., a peristaltic pump) disposed in the conduit.

Also provided are integrated and continuous processes for manufacturinga therapeutic protein drug substance that include: (a) providing aliquid culture medium including a recombinant therapeutic protein thatis substantially free of cells, where the liquid culture medium is fedinto a first multi-column chromatography system (MCCS1); (b) capturingsaid recombinant therapeutic protein in the liquid culture medium usingthe MCCS1, where the eluate of the MCCS1 including the recombinanttherapeutic protein is continuously fed into a second multi-columnchromatography system (MCCS2); (c) purifying and polishing therecombinant therapeutic protein using the MCCS2, wherein the eluate fromthe MCCS2 is a fluid including the recombinant therapeutic protein; (d)processing the fluid including the recombinant therapeutic protein usinga method of any methods described herein; where the process isintegrated and runs continuously from said liquid culture medium to thecollected fluid or filtrate including the recombinant therapeuticprotein, where the collected fluid or filtrate is the therapeuticprotein drug substance. In some embodiments of these processes, theMCCS1 performs at least two different unit operations. In someembodiments of these processes, the MCCS1 performs the unit operationsof capturing the recombinant therapeutic protein and inactivatingviruses. In some embodiments of these processes, the use of the MCCS1 orthe MCCS2, or both, involves column switching. In some embodiments ofthese processes, the MCCS1 and/or MCCS2 utilizes at least twochromatography columns. In some embodiments of these processes, theMCCS1 and/or MCCS2 utilizes at least two chromatographic membranes. Insome embodiments of these processes, the MCCS1 and/or MCCS2 utilizes atleast one chromatography column and at least one chromatographicmembrane. In some embodiments of these processes, the MCCS1 is a firstperiodic counter current chromatography system (PCCS1). In someembodiments of these processes, the PCCS1 includes a four-column PCCS.In some embodiments of these processes, three of the four columns in thefour-column PCCS perform the unit operation of capturing the recombinanttherapeutic protein from the liquid culture medium. In some embodimentsof these processes, the capturing is performed using affinitychromatography, cation exchange chromatography, anion exchangechromatography, or molecular sieve chromatography. In some embodimentsof these processes, the affinity chromatography is performed with acapture mechanism selected from the group of: protein A-binding capturemechanism, substrate-binding capture mechanism, antibody- or antibodyfragment-binding capture mechanism, aptamer-binding capture mechanism,and cofactor-binding capture mechanism. In some embodiments of theseprocesses, the affinity chromatography is performed with a proteinA-binding capture mechanism, and the recombinant therapeutic protein isan antibody or an antibody fragment. In some embodiments of theseprocesses, the eluate containing the recombinant therapeutic proteinfrom three of the four columns in the four-column PCCS is fed into thefourth column of the four-column PCCS. In some embodiments of theseprocesses, the fourth column of the four-column PCCS performs the unitoperation of inactivating viruses by holding the eluate containingrecombinant therapeutic protein at a low pH for viral inactivation. Insome embodiments of these processes, the fourth column of thefour-column PCCS holds the eluate containing the recombinant protein ata low pH for viral inactivation for a period of about 10 minutes toabout 1.5 hours.

In some embodiments of these processes, the MCCS2 is a second periodiccounter current chromatography system (PCCS2). Some embodiments of theseprocesses further include adjusting the pH of the eluate from the fourthcolumn of the four-column PCCS using an in-line buffer adjustmentreservoir before the eluate from the fourth column of the four-columnPCCS is fed into the PCCS2. In some embodiments of these processes, thePCCS2 includes three chromatography columns and a chromatographicmembrane. In some embodiments of these processes, the threechromatographic columns in the PCCS2 perform the unit operation ofpurifying the recombinant therapeutic protein from the eluate of thePCCS1 through cation or anion exchange chromatography. In someembodiments of these processes, the eluate from the three chromatographycolumns in the PCCS2 is fed into the chromatographic membrane in thePCCS2. In some embodiments of these processes, the chromatographicmembrane in the PCCS2 performs the unit operation of polishing therecombinant therapeutic protein present in the eluate from the threechromatographic columns in the PCCS2 through cation or anion exchangechromatography. In some embodiments of these processes, thechromatographic membrane in the PCCS2 performs the unit operation ofpolishing through cation exchange chromatography. In some embodiments ofthese processes, the flow through and wash of the chromatographicmembrane includes the recombinant therapeutic protein. Some embodimentsof these processes further include adjusting the ionic concentration ofthe eluate from the three columns in the PCCS2 using in-line bufferadjustment before the eluate from the three columns in the PCCS2 is fedinto the chromatographic membrane in the PCCS2. Some embodiments ofthese processes further include the use of a break tank between thePCCS1 and the PCCS2. Some embodiments of these processes further includefiltering the eluate from the PCCS1 before it is fed into the PCCS2.Some embodiments of these processes further include formulating thetherapeutic protein drug substance into a pharmaceutical composition. Insome embodiments of any of methods or processes described herein, therecombinant therapeutic protein is an antibody or antibody fragment, anenzyme, an engineered protein, or an immunogenic protein or proteinfragment.

As used herein, the word “a” before a noun represents one or more of theparticular noun. For example, the phrase “a mammalian cell” represents“one or more mammalian cells.”

The terms “tangential flow filtration unit” or “TFF unit” are art-knownand mean a device that includes at least one housing (such as acylinder) and at least one cross-flow (tangential) filter positioned inthe housing such that a large portion of the filter's surface ispositioned parallel to the flow of a fluid (e.g., a cell culture)through the unit. TFF units are well-known in the art and arecommercially available. The housing can include a first inlet/outlet anda second inlet/outlet positioned, e.g., to allow fluid to pass throughthe first inlet/outlet, cross the at least one cross-flow filter, andthrough the second inlet/outlet. In some examples, a circuit system caninclude multiple TFF units, e.g., connected in series and/or inparallel. For example, a circuit system that includes two or more TFFunits can include fluid conduits fluidly connecting neighboring pairs ofTFF units in the system. In other examples, a circuit system can includetwo or more sets of two or more TFF units fluidly connected by fluidconduits. Any of the TFF units described herein or known in the art arecapable of receiving fluid in a first flow direction and a second flowdirection.

The terms “tangential flow virus filtration unit” or “TFVF unit” areart-known and mean a device that includes at least one housing (such asa cylinder) and at least one cross-flow (tangential) virus filterpositioned in the housing such that a large portion of the virusfilter's surface is positioned parallel to the flow of a fluid (e.g., acell culture) through the unit. The housing can include a firstinlet/outlet and a second inlet/outlet positioned, e.g., to allow fluidto pass through the first inlet/outlet, cross the at least onecross-flow virus filter, and through the second inlet/outlet. In someexamples, a circuit system can include multiple TFVF units, e.g.,connected in series and/or in parallel. For example, a circuit systemthat includes two or more TFVF units can include fluid conduits fluidlyconnecting neighboring pairs of TFVF units in the system. In otherexamples, a circuit system can include two or more sets of two or moreTFVF units fluidly connected by fluid conduits. Any of the TFVF unitsdescribed herein or known in the art are capable of receiving fluid in afirst flow direction and a second flow direction.

The term “cross-flow filter” or “tangential filter” is art known andmeans a filter that designed such that it can be positioned in a TFF ora TFVF unit such that a large portion of the filter's surface isparallel to the flow (e.g., first and second flow direction) of a fluid(e.g., a fluid including a recombinant therapeutic protein). Forexample, a cross-flow filter can have any shape that allows fortangential flow filtration, e.g., a tubular or rectangular shape.Particularly useful cross-flow filters are designed to result in a lowamount of fluid turbulence or sheer stress in the fluid (e.g., cellculture) when the fluid is flowed (e.g., unidirectionally flowed tobidirectionally flowed) across the surface of the cross-flow filter.Cross-flow filters are commercially available, e.g., from Sartorius,MembraPure, Millipore, and Pall Corporation.

The term “low turbulence pump” or “LTP” is art-known and means a devicethat can move a fluid (e.g., a fluid including a recombinant therapeuticprotein) within a system or circuit in a single direction (e.g., a firstor second flow direction) or reversibly flowing a fluid (e.g., a fluidincluding a recombinant therapeutic protein) in two directions (a firstand second flow direction) within the system without inducing asubstantial amount of sheer stress or fluid turbulence in the fluid(e.g., a fluid including a recombinant therapeutic protein). When a LTPis used to flow a fluid (e.g., a fluid including a recombinanttherapeutic protein) in alternating first and second flow directions,the second flow direction is approximately opposite to that of the firstflow direction. An example of a LTP is a peristaltic pump. Otherexamples of LTPs are known in the art.

The term “mammalian cell” means any cell from or derived from any mammal(e.g., a human, a hamster, a mouse, a green monkey, a rat, a pig, a cow,or a rabbit). For example, a mammalian cell can be an immortalized cell.In some embodiments, the mammalian cell is a differentiated cell. Insome embodiments, the mammalian cell is an undifferentiated cell.Non-limiting examples of mammalian cells are described herein.Additional examples of mammalian cells are known in the art.

The term “substantially free” means a composition (e.g., a liquidculture medium) that is at least or about 90% free (e.g., at least orabout 95%, 96%, 97%, 98%, or at least or about 99% free, or about 100%free) of a specified substance (e.g., a mammalian cell).

The term “0.5× volume” means about 50% of the volume. The term “0.6×volume” means about 60% of the volume. Likewise, 0.7×, 0.8×, 0.9×, and1.0× means about 70%, 80%, 90%, or 100% of the volume, respectively.

The term “culturing” or “cell culturing” means the maintenance orproliferation of a mammalian cell under a controlled set of physicalconditions.

The term “culture of mammalian cells” means a liquid culture mediumcontaining a plurality of mammalian cells that is maintained orproliferated under a controlled set of physical conditions.

The term “liquid culture medium” means a fluid that contains sufficientnutrients to allow a cell (e.g., a mammalian cell) to grow orproliferate in vitro. For example, a liquid culture medium can containone or more of: amino acids (e.g., 20 amino acids), a purine (e.g.,hypoxanthine), a pyrimidine (e.g., thymidine), choline, inositol,thiamine, folic acid, biotin, calcium, niacinamide, pyridoxine,riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium,glucose, sodium, potassium, iron, copper, zinc, and sodium bicarbonate.In some embodiments, a liquid culture medium can contain serum from amammal. In some embodiments, a liquid culture medium does not containserum or another extract from a mammal (a defined liquid culturemedium). In some embodiments, a liquid culture medium can contain tracemetals, a mammalian growth hormone, and/or a mammalian growth factor.Another example of liquid culture medium is minimal medium (e.g., amedium containing only inorganic salts, a carbon source, and water).Non-limiting examples of liquid culture medium are described herein.Additional examples of liquid culture medium are known in the art andare commercially available. A liquid culture medium can contain anydensity of mammalian cells. For example, as used herein, a volume ofliquid culture medium removed from a bioreactor can be substantiallyfree of mammalian cells.

The term “animal-derived component free liquid culture medium” means aliquid culture medium that does not contain any components (e.g.,proteins or serum) derived from a mammal.

The term “serum-free liquid culture medium” means a liquid culturemedium that does not contain a mammalian serum.

The term “serum-containing liquid culture medium” means a liquid culturemedium that contains a mammalian serum.

The term “chemically-defined liquid culture medium” is a term of art andmeans a liquid culture medium in which all of the chemical componentsare known. For example, a chemically-defined liquid culture medium doesnot contain fetal bovine serum, bovine serum albumin, or human serumalbumin, as these preparations typically contain a complex mix ofalbumins and lipids.

The term “protein-free liquid culture medium” means a liquid culturemedium that does not contain any protein (e.g., any detectable protein).

The term “agitation” means stirring or otherwise moving a portion ofliquid culture medium in a bioreactor. This is performed in order to,e.g., increase the dissolved 02 concentration in the liquid culturemedium in a bioreactor. Agitation can be performed using any art knownmethod, e.g., an instrument or propeller. Exemplary devices and methodsthat can be used to perform agitation of a portion of the liquid culturemedium in a bioreactor are known in the art.

The term “therapeutic protein drug substance” means a recombinantprotein (e.g., an immunoglobulin, protein fragment, engineered protein,or enzyme) that has been sufficiently purified or isolated fromcontaminating proteins, lipids, and nucleic acids (e.g., contaminatingproteins, lipids, and nucleic acids present in a liquid culture mediumor from a host cell (e.g., from a mammalian, yeast, or bacterial hostcell)) and biological contaminants (e.g., viral and bacterialcontaminants), and can be formulated into a pharmaceutical agent withoutany further substantial purification and/or decontamination step.

The term “integrated process” means a process which is performed usingstructural elements that function cooperatively to achieve a specificresult (e.g., the generation of a therapeutic protein drug substancefrom a liquid culture medium).

The term “continuous process” means a process which continuously feedsfluid through at least a part of the system. For example, in any of theexemplary continuous biological manufacturing systems described herein,a liquid culture medium containing a recombinant therapeutic protein iscontinuously fed into the system while it is in operation and atherapeutic protein drug substance is fed out of the system. In anotherexample, a continuous process is a process which continuously feeds aliquid culture medium containing a recombinant therapeutic protein froma bioreactor through a first MCCS. Another example of a continuousprocess is a process which continuously feeds a liquid culture mediumcontaining a recombinant therapeutic protein from a bioreactor through afirst and second MCCS. Additional examples include a process whichcontinuously feeds a liquid culture medium containing a recombinanttherapeutic protein through a first MCCS, a process that continuouslyfeeds a liquid culture medium containing a recombinant therapeuticprotein through a first and second MCCS, or a process that continuouslyfeeds a fluid containing a recombinant therapeutic protein through asecond MCCS.

The term “immunoglobulin” means a polypeptide containing an amino acidsequence of at least 15 amino acids (e.g., at least 20, 30, 40, 50, 60,70, 80, 90, or 100 amino acids) of an immunoglobulin protein (e.g., avariable domain sequence, a framework sequence, or a constant domainsequence). The immunoglobulin may, for example, include at least 15amino acids of a light chain immunoglobulin, e.g., at least 15 aminoacids of a heavy chain immunoglobulin. The immunoglobulin may be anisolated antibody (e.g., an IgG, IgE, IgD, IgA, or IgM). Theimmunoglobulin may be a subclass of IgG (e.g., IgG1, IgG2, IgG3, orIgG4). The immunoglobulin may be an antibody fragment, e.g., a Fabfragment, a F(ab′)₂ fragment, or an scFv fragment. The immunoglobulinmay also be a bi-specific antibody or a tri-specific antibody, or adimer, trimer, or multimer antibody, or a diabody, an Affibody®, or aNanobody®. The immunoglobulin can also be an engineered proteincontaining at least one immunoglobulin domain (e.g., a fusion protein).Non-limiting examples of immunoglobulins are described herein andadditional examples of immunoglobulins are known in the art.

The term “protein fragment” or “polypeptide fragment” means a portion ofa polypeptide sequence that is at least or about 4 amino acids, at leastor about 5 amino acids, at least or about 6 amino acids, at least orabout 7 amino acids, at least or about 8 amino acids, at least or about9 amino acids, at least or about 10 amino acids, at least or about 11amino acids, at least or about 12 amino acids, at least or about 13amino acids, at least or about 14 amino acids, at least or about 15amino acids, at least or about 16 amino acids, at least or about 17amino acids, at least or about 18 amino acids, at least or about 19amino acids, or at least or about 20 amino acids in length, or more than20 amino acids in length. A recombinant protein fragment can be producedusing any of the processes described herein.

The term “engineered protein” means a polypeptide that is not naturallyencoded by an endogenous nucleic acid present within an organism (e.g.,a mammal). Examples of engineered proteins include enzymes (e.g., withone or more amino acid substitutions, deletions, insertions, oradditions that result in an increase in stability and/or catalyticactivity of the engineered enzyme), fusion proteins, antibodies (e.g.,divalent antibodies, trivalent antibodies, or a diabody), andantigen-binding proteins that contain at least one recombinantscaffolding sequence.

The term “multi-column chromatography system” or “MCCS” means a systemof a total of two or more interconnected or switching chromatographycolumns and/or chromatographic membranes. A non-limiting example of amulti-column chromatography system is a periodic counter currentchromatography system (PCC) containing a total of two or moreinterconnected or switching chromatography columns and/orchromatographic membranes. Additional examples of multi-columnchromatography systems are described herein and are known in the art.

The term “capturing” means a step performed to partially purify orisolate (e.g., at least or about 5%, e.g., at least or about 10%, 15%,20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, orat least or about 95% pure by weight), concentrate, and stabilize arecombinant therapeutic protein from one or more other componentspresent in a liquid culture medium or a diluted liquid culture medium(e.g., culture medium proteins or one or more other components (e.g.,DNA, RNA, or other proteins) present in or secreted from a mammaliancell). Typically, capturing is performed using a resin that binds arecombinant therapeutic protein (e.g., through the use of affinitychromatography). Non-limiting methods for capturing a recombinanttherapeutic protein from a liquid culture medium or diluted liquidculture medium are described herein and others are known in the art. Arecombinant therapeutic protein can be captured from a liquid culturemedium using at least one chromatography column and/or chromatographicmembrane (e.g., any of the chromatography columns and/or chromatographicmembranes described herein).

The term “purifying” means a step performed to isolate a recombinanttherapeutic protein from one or more other impurities (e.g., bulkimpurities) or components present in a fluid containing a recombinanttherapeutic protein (e.g., liquid culture medium proteins or one or moreother components (e.g., DNA, RNA, other proteins, endotoxins, viruses,etc.) present in or secreted from a mammalian cell). For example,purifying can be performed during or after an initial capturing step.Purification can be performed using a resin, membrane, or any othersolid support that binds either a recombinant therapeutic protein orcontaminants (e.g., through the use of affinity chromatography,hydrophobic interaction chromatography, anion or cation exchangechromatography, or molecular sieve chromatography). A recombinanttherapeutic protein can be purified from a fluid containing therecombinant therapeutic protein using at least one chromatography columnand/or chromatographic membrane (e.g., any of the chromatography columnsor chromatographic membranes described herein).

The term “polishing” is a term of art and means a step performed toremove remaining trace or small amounts of contaminants or impuritiesfrom a fluid containing a recombinant therapeutic protein that is closeto a final desired purity. For example, polishing can be performed bypassing a fluid containing the recombinant therapeutic protein through achromatographic column(s) or membrane absorber(s) that selectively bindsto either the target recombinant therapeutic protein or small amounts ofcontaminants or impurities present in a fluid containing a recombinanttherapeutic protein. In such an example, the eluate/filtrate of thechromatographic column(s) or membrane absorber(s) contains therecombinant therapeutic protein.

The term “eluate/filtrate” is a term of art and means a fluid that isemitted from a chromatography column or chromatographic membrane thatcontains a detectable amount of a recombinant therapeutic protein.

The term “filtering” means the removal of at least part of (e.g., atleast 80%, 90%, 95%, 96%, 97%, 98%, or 99%) undesired biologicalcontaminants (e.g., a mammalian cell, bacteria, yeast cells, viruses, ormycobacteria) and/or particulate matter (e.g., precipitated proteins)from a liquid (e.g., a liquid culture medium or fluid present in any ofthe systems or processes described herein).

The term “secreted protein” or “secreted recombinant protein” means aprotein (e.g., a recombinant protein) that originally contained at leastone secretion signal sequence when it is translated within a mammaliancell, and through, at least in part, enzymatic cleavage of the secretionsignal sequence in the mammalian cell, is secreted at least partiallyinto the extracellular space (e.g., a liquid culture medium). Skilledpractitioners will appreciate that a “secreted” protein need notdissociate entirely from the cell to be considered a secreted protein.

The term “perfusion bioreactor” means a bioreactor containing aplurality of cells (e.g., mammalian cells) in a first liquid culturemedium, wherein the culturing of the cells present in the bioreactorincludes periodic or continuous removal of the first liquid culturemedium and at the same time or shortly thereafter adding substantiallythe same volume of a second liquid culture medium to the bioreactor. Insome examples, there is an incremental change (e.g., increase ordecrease) in the volume of the first liquid culture medium removed andadded over incremental periods (e.g., an about 24-hour period, a periodof between about 1 minute and about 24-hours, or a period of greaterthan 24 hours) during the culturing period (e.g., the culture mediumrefeed rate on a daily basis). The fraction of media removed andreplaced each day can vary depending on the particular cells beingcultured, the initial seeding density, and the cell density at aparticular time. “RV” or “reactor volume” means the volume of theculture medium present at the beginning of the culturing process (e.g.,the total volume of the culture medium present after seeding).

The term “fed-batch bioreactor” is a term of art and means a bioreactorcontaining a plurality of cells (e.g., mammalian cells) in a firstliquid culture medium, wherein the culturing of the cells present in thebioreactor includes the periodic or continuous addition of a secondliquid culture medium to the first liquid culture medium withoutsubstantial or significant removal of the first liquid culture medium orsecond liquid culture medium from the cell culture. The second liquidculture medium can be the same as the first liquid culture medium. Insome examples of fed-batch culture, the second liquid culture medium isa concentrated form of the first liquid culture medium. In some examplesof fed-batch culture, the second liquid culture medium is added as a drypowder.

The term “clarified liquid culture medium” means a liquid culture mediumobtained from a bacterial or yeast cell culture that is substantiallyfree (e.g., at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% free) ofbacteria or yeast cells.

The term “unit operation” is a term of art and means a functional stepthat can be performed in a process of manufacturing a therapeuticprotein drug substance from a liquid culture medium. For example, a unitof operation can be filtering (e.g., removal of contaminant bacteria,yeast viruses, or mycobacteria, and/or particular matter from a fluidcontaining a recombinant therapeutic protein), capturing, epitope tagremoval, purifying, holding or storing, polishing, viral inactivating,adjusting the ionic concentration and/or pH of a fluid containing therecombinant therapeutic protein, and removing unwanted salts.

“Specific productivity rate” or “SPR” is a term of art and as usedherein refers to the mass or enzymatic activity of a recombinanttherapeutic protein produced per mammalian cell per day. The SPR for arecombinant therapeutic antibody is usually measured as mass/cell/day.The SPR for a recombinant therapeutic enzyme is usually measured asunits/cell/day or (units/mass)/cell/day.

“Volume productivity rate” or “VPR” is a term of art and as used hereinrefers to the mass or enzymatic activity of recombinant therapeuticprotein produced per volume of culture (e.g., per L of bioreactor,vessel, or tube volume) per day. The VPR for a recombinant therapeuticantibody is usually measured as mass/L/day. The VPR for a recombinanttherapeutic enzyme is usually measured as units/L/day or mass/L/day.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic showing a two-PCCS manufacturing system connectedwith a perfusion culture bioreactor that results in the continuousmanufacture of a therapeutic protein drug substance. The asteriskindicates an exemplary position where one or both of a circuit systemincluding a tangential flow filtration (TFF) unit (e.g., any of thecircuit systems including a TFF unit described herein) and a circuitsystem including a tangential flow virus filtration (TFVF) unit (e.g.,any of the circuit systems including a TFVF unit described herein) canbe positioned in this exemplary manufacturing system.

FIG. 2 is a schematic showing a manufacturing system connected with aperfusion culture bioreactor that results in the continuous manufactureof a therapeutic protein drug substance. The asterisks indicateexemplary positions where one or both of a circuit system including aTFF unit (e.g., any of the circuit systems including a TFF unitdescribed herein) and a circuit system including a tangential flow virusfiltration (TFVF) unit (e.g., any of the circuit systems including aTFVF unit described herein) can be positions in this exemplarymanufacturing system.

FIG. 3 is a diagram of a PCC system cycle containing threechromatography columns. At the beginning of a cycle, the feed solutionis loaded onto column 1, and the flow through goes to waste untilproduct breakthrough occurs (step 1). At this point, the flow throughfrom column 1 is directed to column 2 to capture the unbound recombinanttherapeutic protein from column 1 (step 2). Once column 1 is fullyloaded, the feed is now directed loaded onto column 2, while column 1 iswashed, eluted, regenerated, and re-equilibrated for the next cycle(steps 3 and 4). Column 2 now goes through steps 3-5, which areidentical to steps 1-3 for column 1. Finally, column 3 goes throughsteps 5-6, the same as columns 1 and 2. Once all three columns havecompleted these steps, the cycle restarts with column 1.

FIG. 4 is a schematic diagram demonstrating the principle of columnswitching based on ΔUV. T1 designates the time when the ΔUV has reacheda pre-determined threshold level. Once this threshold level is reached,the flow through from column 1 is directed onto column 2 rather than tothe waste. T2 designates the time when the column has been saturatedwith product. The ΔUV value for both T1 and T2 are process specific.

FIG. 5 is a schematic of the circuit system including a tangential flowfiltration unit during a first period of time, when a fluid including arecombinant therapeutic protein is continuously flowed through into thesystem (“transfer”) and a filtrate not including the recombinanttherapeutic protein is discarded (“permeate”) (left panel) and a graphshowing the mass flux (g/h) over time (h) during the first period oftime (right panel). The recombinant therapeutic protein is retainedwithin the circuit system and is recirculated through the circuit system(“retentate”) during the first period of time.

FIG. 6 is a schematic of the circuit system including a tangential flowfiltration unit during a second period of time, when a diafiltrationmedium is continuously flowed through into the system (“DF buffer”) anda filtrate not including the recombinant therapeutic protein isdiscarded (“permeate”) (left panel) and a graph showing the mass flux(g/h) over time (h) during the second period of time (right panel). Therecombinant therapeutic protein is retained within the circuit systemand is recirculated through the circuit system (“retentate”) during thesecond period of time.

FIG. 7 is a graph illustrating the results of the use of an exemplarycircuit system including a tangential flow filtration unit to performultrafiltration/diafiltration of a fluid including a recombinanttherapeutic protein. The flux (LMH), the fold-concentration of therecombinant therapeutic protein (Fold), the volume of fluid in thecircuit system, the diavolumes, and transmembrane pressure (TMP) of thecircuit system including a tangential flow filtration unit over time areshown.

FIG. 8 is a graph illustrating the results of the use of an exemplarycircuit system including a tangential flow filtration unit to performultrafiltration/diafiltration of a fluid including a recombinanttherapeutic protein. The flux (LMH), the fold-concentration of therecombinant therapeutic protein (Fold), the volume of fluid in thecircuit system, the diavolumes, and transmembrane pressure (TMP) of thecircuit system including a tangential flow filtration unit over time areshown.

FIG. 9 is a graph illustrating the results of the use of an exemplarycircuit system including a tangential flow filtration unit to performultrafiltration/diafiltration of a fluid including a recombinanttherapeutic protein. The flux (LMH), the fold-concentration of therecombinant therapeutic protein (Fold), the volume of fluid in thecircuit system, the diavolumes, and transmembrane pressure (TMP) of thecircuit system including a tangential flow filtration unit over time areshown.

FIG. 10 is a schematic of the circuit system including a tangential flowvirus filtration unit, when a fluid including a recombinant therapeuticprotein is continuously flowed into the system (“from UF”) and afiltrate including the recombinant therapeutic protein is continuouslycollected (“VF product”).

FIG. 11 is a schematic showing an exemplary circuit system including atangential flow virus filtration unit.

FIG. 12 is a graph showing the data obtained from the use of a circuitsystem including a tangential flow virus filtration unit to performvirus filtration. The black line represents the pressure (psi) and thegray line represents the mass of the permeate (kg) over time.

FIG. 13 is a graph illustrating the results of the use of an exemplarycircuit system including a TFVF unit to perform viral filtration ofMurine Minute Virus (MMV) from a recombinant human antibody (mAb) usinga Planova™ 20N filter operated at 12 psi with a varying recirculationrate from 1-2 mL/min. The mass and pressure profiles of the circuitsystem including a TFVF unit over 2.5 hours are shown.

FIG. 14 is a graph illustrating the results of the use of an exemplarycircuit system including a TFVF unit to perform continuous viralfiltration of Murine Minute Virus (MMV) from a recombinant humanantibody (mAb) using a Planova™ 20N filter operated at 6.6 psi with avarying recirculation rate of 2 mL/min. The mass and pressure profilesof the TFVF unit over 22 hours are shown.

FIG. 15 is a graph illustrating the results of the use of an exemplarycircuit system including a TFVF unit to perform continuous viralfiltration of Murine MinuteVirus (MMV) from a recombinant human antibody(mAb) with refeed using a Planova™ BioEx (3 cm² area) operated at 19.8psi with a varying recirculation rate of 1 mL/min. The mass and pressureprofiles of the TFVF unit over 29 hours are shown.

FIG. 16 is a graph illustrating the results of the use of an exemplarycircuit system including a TFVF unit to perform continuous viralfiltration of Murine Minute Virus (MMV) from a recombinant humanantibody (mAb) with refeed using a Planova™ BioEx (10 cm² area) operatedat 19.8 psi with a varying recirculation rate of 3 mL/min. The mass andpressure profiles of the TFVF unit over 29 hours are shown.

DETAILED DESCRIPTION

Provided herein are methods of processing a fluid including arecombinant therapeutic protein that include (a) providing a circuitsystem including (i) a tangential flow filtration (TFF) unit havingfirst and second inlets, and (ii) a conduct in fluid communicationbetween the first and second inlets, including at least one port forflowing fluid into or out of, or both, of the system, where the systemis configured such that fluid can be flowed through the conduit and theTFF unit, and filtrate not including the recombinant therapeutic proteincan be collected from the TFF unit; (b) continuously flowing a fluidincluding a recombinant therapeutic protein into the circuit systemthrough one of the at least one port, and discarding filtrate notincluding the recombinant therapeutic protein for a first period oftime; (c) continuously flowing a diafiltration medium into the circuitsystem through one of the at least one port, and discarding filtrate notincluding the recombinant therapeutic protein for a second period oftime; and (d) collecting a fluid including the recombinant therapeuticprotein that is present in the circuit system after the first and secondtime periods through one of the at least one port. Also provided aremethods of processing a fluid including a recombinant therapeuticprotein that include: (a) providing a circuit system including (i) atangential flow virus filtration (TFVF) unit having first and secondinlets, and (ii) a conduit in fluid communication between the first andsecond inlets of the TFVF unit, including at least one port for flowingfluid into or out of, or both, of the system, where the system isconfigured such that fluid can be flowed through the conduit and theTFVF unit, and filtrate including the recombinant therapeutic proteincan be collected from the TFVF unit; and (b) continuously flowing afluid including a recombinant therapeutic protein into the circuitsystem through one of the at least one port, and collecting filtratecomprising the recombinant therapeutic protein from the TFVF unit for aperiod of time. As can be appreciated by those skilled in the art, someembodiments of these methods can further include recirculating any fluidincluding the recombinant therapeutic protein in the conduit of thecircuit system including a TFVF unit that did not pass through the oneor more tangential virus filters in the TFVF unit (e.g., acts as abuffer when a new volume of a fluid including the recombinanttherapeutic protein is flowed into the conduit of the circuit systemincluding a TFVF unit).

Also provided are integrated and continuous processes for manufacturinga therapeutic protein drug substance that include: (a) providing aliquid culture medium including a recombinant therapeutic protein thatis substantially free of cells, wherein the liquid culture medium is fedinto a first multi-column chromatography system (MCCS1); (b) capturingthe recombinant therapeutic protein in the liquid culture medium usingthe MCCS1, where the eluate of the MCCS1 containing the recombinanttherapeutic protein is continuously fed into a second multi-columnchromatography system (MCCS2); (c) purifying and polishing therecombinant therapeutic protein using the MCCS2, where the eluate fromthe MCCS2 is a fluid including the recombinant therapeutic protein; (d)processing the fluid including the recombinant therapeutic protein usinga method of any of the methods described herein, where the process isintegrated and runs continuously from said liquid culture medium to thecollected fluid or filtrate comprising the recombinant therapeuticprotein, wherein the collected fluid or filtrate is the therapeuticprotein drug substance.

Methods of Processing a Fluid Including a Recombinant TherapeuticProtein that Include the Use of a Circuit System Including a TFF Unit

Provided herein are methods of processing a fluid including arecombinant therapeutic protein that include: providing a circuit systemincluding: (a) a tangential flow filtration (TFF) unit having first andsecond inlets, and (ii) a conduit in fluid communication between thefirst and second inlets, including at least one port for flowing fluidinto or out of, or both, of the system; (b) continuously flowing a fluidincluding a recombinant therapeutic protein into the circuit systemthrough one of the at least one port, and discarding filtrate notincluding the recombinant therapeutic protein for a first period oftime; (c) continuously flowing a diafiltration medium into the circuitsystem through one of the at least one port, and discarding filtrate notincluding the recombinant therapeutic protein for a second period oftime; and (d) collecting a fluid including the recombinant therapeuticprotein that is present in the circuit system after the first and secondtime points through one of the at least one port. As can be appreciatedby those skilled in the art, some embodiments of these methods includerecirculating any fluid including the recombinant therapeutic protein inthe conduit of the circuit system including a TFF unit that did not passthrough the one or more tangential filters in the TFF unit (e.g., actsas a buffer when a new volume of a fluid including the recombinanttherapeutic protein and/or a diafiltration medium is flowed into theconduit of the circuit system including a TFF unit).

In some embodiments, the TFF unit includes one, two, three, four, five,six, seven, eight, nine, ten, eleven, or twelve tangential filters. Insome examples of these methods, the TFF unit can include one or moretangential filter(s) having a surface area of about 0.05 cm² to about 50cm², about 48 cm², about 46 cm², about 44 cm², about 42 cm², about 40cm², about 38 cm², about 36 cm², about 34 cm², about 32 cm², about 30cm², about 28 cm², about 26 cm², about 24 cm², about 22 cm², about 20cm², about 18 cm², about 16 cm², about 14 cm², about 12 cm², about 10cm², about 9.5 cm², about 9.0 cm², about 8.5 cm², about 8.0 cm², about7.5 cm², about 7.0 cm², about 6.5 cm², about 6.0 cm², about 5.5 cm²,about 5.0 cm², about 4.5 cm², about 4.0 cm², about 3.5 cm², about 3.0cm², about 2.5 cm², about 2.0 cm², about 1.5 cm², about 1.0 cm², about0.5 cm², or about 0.1 cm²; about 0.1 cm² to about 50 cm², about 48 cm²,about 46 cm², about 44 cm², about 42 cm², about 40 cm², about 38 cm²,about 36 cm², about 34 cm², about 32 cm², about 30 cm², about 28 cm²,about 26 cm², about 24 cm², about 22 cm², about 20 cm², about 18 cm²,about 16 cm², about 14 cm², about 12 cm², about 10 cm², about 9.5 cm²,about 9.0 cm², about 8.5 cm², about 8.0 cm², about 7.5 cm², about 7.0cm², about 6.5 cm², about 6.0 cm², about 5.5 cm², about 5.0 cm², about4.5 cm², about 4.0 cm², about 3.5 cm², about 3.0 cm², about 2.5 cm²,about 2.0 cm², about 1.5 cm², about 1.0 cm², or about 0.5 cm²; about 0.5cm² to about 50 cm², about 48 cm², about 46 cm², about 44 cm², about 42cm², about 40 cm², about 38 cm², about 36 cm², about 34 cm², about 32cm², about 30 cm², about 28 cm², about 26 cm², about 24 cm², about 22cm², about 20 cm², about 18 cm², about 16 cm², about 14 cm², about 12cm², about 10 cm², about 9.5 cm², about 9.0 cm², about 8.5 cm², about8.0 cm², about 7.5 cm², about 7.0 cm², about 6.5 cm², about 6.0 cm²,about 5.5 cm², about 5.0 cm², about 4.5 cm², about 4.0 cm², about 3.5cm², about 3.0 cm², about 2.5 cm², about 2.0 cm², about 1.5 cm², orabout 1.0 cm²; about 1.0 cm² to about 50 cm², about 48 cm², about 46cm², about 44 cm², about 42 cm², about 40 cm², about 38 cm², about 36cm², about 34 cm², about 32 cm², about 30 cm², about 28 cm², about 26cm², about 24 cm², about 22 cm², about 20 cm², about 18 cm², about 16cm², about 14 cm², about 12 cm², about 10 cm², about 9.5 cm², about 9.0cm², about 8.5 cm², about 8.0 cm², about 7.5 cm², about 7.0 cm², about6.5 cm², about 6.0 cm², about 5.5 cm², about 5.0 cm², about 4.5 cm²,about 4.0 cm², about 3.5 cm², about 3.0 cm², about 2.5 cm², about 2.0cm², about 1.5 cm²; about 1.5 cm² to about 50 cm², about 48 cm², about46 cm², about 44 cm², about 42 cm², about 40 cm², about 38 cm², about 36cm², about 34 cm², about 32 cm², about 30 cm², about 28 cm², about 26cm², about 24 cm², about 22 cm², about 20 cm², about 18 cm², about 16cm², about 14 cm², about 12 cm², about 10 cm², about 9.5 cm², about 9.0cm², about 8.5 cm², about 8.0 cm², about 7.5 cm², about 7.0 cm², about6.5 cm², about 6.0 cm², about 5.5 cm², about 5.0 cm², about 4.5 cm²,about 4.0 cm², about 3.5 cm², about 3.0 cm², about 2.5 cm², or about 2.0cm²; about 2.0 cm² to about 50 cm², about 48 cm², about 46 cm², about 44cm², about 42 cm², about 40 cm², about 38 cm², about 36 cm², about 34cm², about 32 cm², about 30 cm², about 28 cm², about 26 cm², about 24cm², about 22 cm², about 20 cm², about 18 cm², about 16 cm², about 14cm², about 12 cm², about 10 cm², about 9.5 cm², about 9.0 cm², about 8.5cm², about 8.0 cm², about 7.5 cm², about 7.0 cm², about 6.5 cm², about6.0 cm², about 5.5 cm², about 5.0 cm², about 4.5 cm², about 4.0 cm²,about 3.5 cm², about 3.0 cm², or about 2.5 cm²; about 2.5 cm² to about50 cm², about 48 cm², about 46 cm², about 44 cm², about 42 cm², about 40cm², about 38 cm², about 36 cm², about 34 cm², about 32 cm², about 30cm², about 28 cm², about 26 cm², about 24 cm², about 22 cm², about 20cm², about 18 cm², about 16 cm², about 14 cm², about 12 cm², about 10cm², about 9.5 cm², about 9.0 cm², about 8.5 cm², about 8.0 cm², about7.5 cm², about 7.0 cm², about 6.5 cm², about 6.0 cm², about 5.5 cm²,about 5.0 cm², about 4.5 cm², about 4.0 cm², about 3.5 cm², or about 3.0cm²; about 3.0 cm² to about 50 cm², about 48 cm², about 46 cm², about 44cm², about 42 cm², about 40 cm², about 38 cm², about 36 cm², about 34cm², about 32 cm², about 30 cm², about 28 cm², about 26 cm², about 24cm², about 22 cm², about 20 cm², about 18 cm², about 16 cm², about 14cm², about 12 cm², about 10 cm², about 9.5 cm², about 9.0 cm², about 8.5cm², about 8.0 cm², about 7.5 cm², about 7.0 cm², about 6.5 cm², about6.0 cm², about 5.5 cm², about 5.0 cm², about 4.5 cm², about 4.0 cm², orabout 3.5 cm²; about 3.5 cm² to about 50 cm², about 48 cm², about 46cm², about 44 cm², about 42 cm², about 40 cm², about 38 cm², about 36cm², about 34 cm², about 32 cm², about 30 cm², about 28 cm², about 26cm², about 24 cm², about 22 cm², about 20 cm², about 18 cm², about 16cm², about 14 cm², about 12 cm², about 10 cm², about 9.5 cm², about 9.0cm², about 8.5 cm², about 8.0 cm², about 7.5 cm², about 7.0 cm², about6.5 cm², about 6.0 cm², about 5.5 cm², about 5.0 cm², about 4.5 cm², orabout 4.0 cm²; about 4.0 cm² to about 50 cm², about 48 cm², about 46cm², about 44 cm², about 42 cm², about 40 cm², about 38 cm², about 36cm², about 34 cm², about 32 cm², about 30 cm², about 28 cm², about 26cm², about 24 cm², about 22 cm², about 20 cm², about 18 cm², about 16cm², about 14 cm², about 12 cm², about 10 cm², about 9.5 cm², about 9.0cm², about 8.5 cm², about 8.0 cm², about 7.5 cm², about 7.0 cm², about6.5 cm², about 6.0 cm², about 5.5 cm², about 5.0 cm², or about 4.5 cm²;about 4.5 cm² to about 50 cm², about 48 cm², about 46 cm², about 44 cm²,about 42 cm², about 40 cm², about 38 cm², about 36 cm², about 34 cm²,about 32 cm², about 30 cm², about 28 cm², about 26 cm², about 24 cm²,about 22 cm², about 20 cm², about 18 cm², about 16 cm², about 14 cm²,about 12 cm², about 10 cm², about 9.5 cm², about 9.0 cm², about 8.5 cm²,about 8.0 cm², about 7.5 cm², about 7.0 cm², about 6.5 cm², about 6.0cm², about 5.5 cm², or about 5.0 cm²; about 5.0 cm² to about 50 cm²,about 48 cm², about 46 cm², about 44 cm², about 42 cm², about 40 cm²,about 38 cm², about 36 cm², about 34 cm², about 32 cm², about 30 cm²,about 28 cm², about 26 cm², about 24 cm², about 22 cm², about 20 cm²,about 18 cm², about 16 cm², about 14 cm², about 12 cm², about 10 cm²,about 9.5 cm², about 9.0 cm², about 8.5 cm², about 8.0 cm², about 7.5cm², about 7.0 cm², about 6.5 cm², about 6.0 cm², or about 5.5 cm²;about 5.5 cm² to about 50 cm², about 48 cm², about 46 cm², about 44 cm²,about 42 cm², about 40 cm², about 38 cm², about 36 cm², about 34 cm²,about 32 cm², about 30 cm², about 28 cm², about 26 cm², about 24 cm²,about 22 cm², about 20 cm², about 18 cm², about 16 cm², about 14 cm²,about 12 cm², about 10 cm², about 9.5 cm², about 9.0 cm², about 8.5 cm²,about 8.0 cm², about 7.5 cm², about 7.0 cm², about 6.5 cm², or about 6.0cm²; about 6.0 cm² to about 50 cm², about 48 cm², about 46 cm², about 44cm², about 42 cm², about 40 cm², about 38 cm², about 36 cm², about 34cm², about 32 cm², about 30 cm², about 28 cm², about 26 cm², about 24cm², about 22 cm², about 20 cm², about 18 cm², about 16 cm², about 14cm², about 12 cm², about 10 cm², about 9.5 cm², about 9.0 cm², about 8.5cm², about 8.0 cm², about 7.5 cm², about 7.0 cm², or about 6.5 cm²;about 6.5 cm² to about 50 cm², about 48 cm², about 46 cm², about 44 cm²,about 42 cm², about 40 cm², about 38 cm², about 36 cm², about 34 cm²,about 32 cm², about 30 cm², about 28 cm², about 26 cm², about 24 cm²,about 22 cm², about 20 cm², about 18 cm², about 16 cm², about 14 cm²,about 12 cm², about 10 cm², about 9.5 cm², about 9.0 cm², about 8.5 cm²,about 8.0 cm², about 7.5 cm², or about 7.0 cm²; about 7.0 cm² to about50 cm², about 48 cm², about 46 cm², about 44 cm², about 42 cm², about 40cm², about 38 cm², about 36 cm², about 34 cm², about 32 cm², about 30cm², about 28 cm², about 26 cm², about 24 cm², about 22 cm², about 20cm², about 18 cm², about 16 cm², about 14 cm², about 12 cm², about 10cm², about 9.5 cm², about 9.0 cm², about 8.5 cm², about 8.0 cm², orabout 7.5 cm²; about 7.5 cm² to about 50 cm², about 48 cm², about 46cm², about 44 cm², about 42 cm², about 40 cm², about 38 cm², about 36cm², about 34 cm², about 32 cm², about 30 cm², about 28 cm², about 26cm², about 24 cm², about 22 cm², about 20 cm², about 18 cm², about 16cm², about 14 cm², about 12 cm², about 10 cm², about 9.5 cm², about 9.0cm², about 8.5 cm², or about 8.0 cm²; about 8.0 cm² to about 50 cm²,about 48 cm², about 46 cm², about 44 cm², about 42 cm², about 40 cm²,about 38 cm², about 36 cm², about 34 cm², about 32 cm², about 30 cm²,about 28 cm², about 26 cm², about 24 cm², about 22 cm², about 20 cm²,about 18 cm², about 16 cm², about 14 cm², about 12 cm², about 10 cm²,about 9.5 cm², about 9.0 cm², or about 8.5 cm²; about 8.5 cm² to about50 cm², about 48 cm², about 46 cm², about 44 cm², about 42 cm², about 40cm², about 38 cm², about 36 cm², about 34 cm², about 32 cm², about 30cm², about 28 cm², about 26 cm², about 24 cm², about 22 cm², about 20cm², about 18 cm², about 16 cm², about 14 cm², about 12 cm², about 10cm², about 9.5 cm², or about 9.0 cm²; about 9.0 cm² to about 50 cm²,about 48 cm², about 46 cm², about 44 cm², about 42 cm², about 40 cm²,about 38 cm², about 36 cm², about 34 cm², about 32 cm², about 30 cm²,about 28 cm², about 26 cm², about 24 cm², about 22 cm², about 20 cm²,about 18 cm², about 16 cm², about 14 cm², about 12 cm², about 10 cm², orabout 9.5 cm²; about 9.5 cm² to about 50 cm², about 48 cm², about 46cm², about 44 cm², about 42 cm², about 40 cm², about 38 cm², about 36cm², about 34 cm², about 32 cm², about 30 cm², about 28 cm², about 26cm², about 24 cm², about 22 cm², about 20 cm², about 18 cm², about 16cm², about 14 cm², about 12 cm², or about 10 cm²; about 10 cm² to about50 cm², about 48 cm², about 46 cm², about 44 cm², about 42 cm², about 40cm², about 38 cm², about 36 cm², about 34 cm², about 32 cm², about 30cm², about 28 cm², about 26 cm², about 24 cm², about 22 cm², about 20cm², about 18 cm², about 16 cm², about 14 cm², or about 12 cm²; about 12cm² to about 50 cm², about 48 cm², about 46 cm², about 44 cm², about 42cm², about 40 cm², about 38 cm², about 36 cm², about 34 cm², about 32cm², about 30 cm², about 28 cm², about 26 cm², about 24 cm², about 22cm², about 20 cm², about 18 cm², about 16 cm², or about 14 cm²; about 14cm² to about 50 cm², about 48 cm², about 46 cm², about 44 cm², about 42cm², about 40 cm², about 38 cm², about 36 cm², about 34 cm², about 32cm², about 30 cm², about 28 cm², about 26 cm², about 24 cm², about 22cm², about 20 cm², about 18 cm², or about 16 cm²; about 16 cm² to about50 cm², about 48 cm², about 46 cm², about 44 cm², about 42 cm², about 40cm², about 38 cm², about 36 cm², about 34 cm², about 32 cm², about 30cm², about 28 cm², about 26 cm², about 24 cm², about 22 cm², about 20cm², or about 18 cm²; about 18 cm² to about 50 cm², about 48 cm², about46 cm², about 44 cm², about 42 cm², about 40 cm², about 38 cm², about 36cm², about 34 cm², about 32 cm², about 30 cm², about 28 cm², about 26cm², about 24 cm², about 22 cm², or about 20 cm²; about 20 cm² to about50 cm², about 48 cm², about 46 cm², about 44 cm², about 42 cm², about 40cm², about 38 cm², about 36 cm², about 34 cm², about 32 cm², about 30cm², about 28 cm², about 26 cm², about 24 cm², or about 22 cm²; about 22cm² to about 50 cm², about 48 cm², about 46 cm², about 44 cm², about 42cm², about 40 cm², about 38 cm², about 36 cm², about 34 cm², about 32cm², about 30 cm², about 28 cm², about 26 cm², or about 24 cm²; about 24cm² to about 50 cm², about 48 cm², about 46 cm², about 44 cm², about 42cm², about 40 cm², about 38 cm², about 36 cm², about 34 cm², about 32cm², about 30 cm², about 28 cm², or about 26 cm²; about 26 cm² to about50 cm², about 48 cm², about 46 cm², about 44 cm², about 42 cm², about 40cm², about 38 cm², about 36 cm², about 34 cm², about 32 cm², about 30cm², or about 28 cm²; about 28 cm² to about 50 cm², about 48 cm², about46 cm², about 44 cm², about 42 cm², about 40 cm², about 38 cm², about 36cm², about 34 cm², about 32 cm², or about 30 cm²; about 30 cm² to about50 cm², about 48 cm², about 46 cm², about 44 cm², about 42 cm², about 40cm², about 38 cm², about 36 cm², about 34 cm², or about 32 cm²; about 32cm² to about 50 cm², about 48 cm², about 46 cm², about 44 cm², about 42cm², about 40 cm², about 38 cm², about 36 cm², or about 34 cm²; about 34cm² to about 50 cm², about 48 cm², about 46 cm², about 44 cm², about 42cm², about 40 cm², about 38 cm², or about 36 cm²; about 36 cm² to about50 cm², about 48 cm², about 46 cm², about 44 cm², about 42 cm², about 40cm², or about 38 cm²; about 38 cm² to about 50 cm², about 48 cm², about46 cm², about 44 cm², about 42 cm², or about 40 cm²; about 40 cm² toabout 50 cm², about 48 cm², about 46 cm², about 44 cm², or about 42 cm²;about 42 cm² to about 50 cm², about 48 cm², about 46 cm², or about 44cm²; about 44 cm² to about 50 cm², about 48 cm², or about 46 cm²; about46 cm² to about 50 cm² or about 48 cm²; or about 48 cm² to about 50 cm².

In some embodiments, the one or more tangential filters in the TFF unitcan have a molecular weight cut-off of about 1 kDa to about 25,000 kDa,about 24,000 kDa, about 22,000 kDa, about 20,000 kDa, about 18,000 kDa,about 16,000 kDa, about 14,000 kDa, about 12,000 kDa, about 10,000 kDa,about 8,000 kDa, about 6,000 kDa, about 4,000 kDa, about 2,000 kDa,about 1,800 kDa, about 1,600 kDa, about 1,400 kDa, about 1,200 kDa,about 1,000 kDa, about 950 kDa, about 900 kDa, about 850 kDa, about 800kDa, about 750 kDa, about 700 kDa, about 650 kDa, about 600 kDa, about550 kDa, about 500 kDa, about 450 kDa, about 400 kDa, about 350 kDa,about 300 kDa, about 250 kDa, about 200 kDa, about 150 kDa, about 100kDa, about 80 kDa, about 60 kDa, about 40 kDa, about 20 kDa, about 10kDa, or about 5 kDa; about 5 kDa to about 25,000 kDa, about 24,000 kDa,about 22,000 kDa, about 20,000 kDa, about 18,000 kDa, about 16,000 kDa,about 14,000 kDa, about 12,000 kDa, about 10,000 kDa, about 8,000 kDa,about 6,000 kDa, about 4,000 kDa, about 2,000 kDa, about 1,800 kDa,about 1,600 kDa, about 1,400 kDa, about 1,200 kDa, about 1,000 kDa,about 950 kDa, about 900 kDa, about 850 kDa, about 800 kDa, about 750kDa, about 700 kDa, about 650 kDa, about 600 kDa, about 550 kDa, about500 kDa, about 450 kDa, about 400 kDa, about 350 kDa, about 300 kDa,about 250 kDa, about 200 kDa, about 150 kDa, about 100 kDa, about 80kDa, about 60 kDa, about 40 kDa, about 20 kDa, or about 10 kDa; about 10kDa to about 25,000 kDa, about 24,000 kDa, about 22,000 kDa, about20,000 kDa, about 18,000 kDa, about 16,000 kDa, about 14,000 kDa, about12,000 kDa, about 10,000 kDa, about 8,000 kDa, about 6,000 kDa, about4,000 kDa, about 2,000 kDa, about 1,800 kDa, about 1,600 kDa, about1,400 kDa, about 1,200 kDa, about 1,000 kDa, about 950 kDa, about 900kDa, about 850 kDa, about 800 kDa, about 750 kDa, about 700 kDa, about650 kDa, about 600 kDa, about 550 kDa, about 500 kDa, about 450 kDa,about 400 kDa, about 350 kDa, about 300 kDa, about 250 kDa, about 200kDa, about 150 kDa, about 100 kDa, about 80 kDa, about 60 kDa, about 40kDa, or about 20 kDa; about 20 kDa to about 25,000 kDa, about 24,000kDa, about 22,000 kDa, about 20,000 kDa, about 18,000 kDa, about 16,000kDa, about 14,000 kDa, about 12,000 kDa, about 10,000 kDa, about 8,000kDa, about 6,000 kDa, about 4,000 kDa, about 2,000 kDa, about 1,800 kDa,about 1,600 kDa, about 1,400 kDa, about 1,200 kDa, about 1,000 kDa,about 950 kDa, about 900 kDa, about 850 kDa, about 800 kDa, about 750kDa, about 700 kDa, about 650 kDa, about 600 kDa, about 550 kDa, about500 kDa, about 450 kDa, about 400 kDa, about 350 kDa, about 300 kDa,about 250 kDa, about 200 kDa, about 150 kDa, about 100 kDa, about 80kDa, about 60 kDa, or about 40 kDa; about 40 kDa to about 25,000 kDa,about 24,000 kDa, about 22,000 kDa, about 20,000 kDa, about 18,000 kDa,about 16,000 kDa, about 14,000 kDa, about 12,000 kDa, about 10,000 kDa,about 8,000 kDa, about 6,000 kDa, about 4,000 kDa, about 2,000 kDa,about 1,800 kDa, about 1,600 kDa, about 1,400 kDa, about 1,200 kDa,about 1,000 kDa, about 950 kDa, about 900 kDa, about 850 kDa, about 800kDa, about 750 kDa, about 700 kDa, about 650 kDa, about 600 kDa, about550 kDa, about 500 kDa, about 450 kDa, about 400 kDa, about 350 kDa,about 300 kDa, about 250 kDa, about 200 kDa, about 150 kDa, about 100kDa, about 80 kDa, or about 60 kDa; about 60 kDa to about 25,000 kDa,about 24,000 kDa, about 22,000 kDa, about 20,000 kDa, about 18,000 kDa,about 16,000 kDa, about 14,000 kDa, about 12,000 kDa, about 10,000 kDa,about 8,000 kDa, about 6,000 kDa, about 4,000 kDa, about 2,000 kDa,about 1,800 kDa, about 1,600 kDa, about 1,400 kDa, about 1,200 kDa,about 1,000 kDa, about 950 kDa, about 900 kDa, about 850 kDa, about 800kDa, about 750 kDa, about 700 kDa, about 650 kDa, about 600 kDa, about550 kDa, about 500 kDa, about 450 kDa, about 400 kDa, about 350 kDa,about 300 kDa, about 250 kDa, about 200 kDa, about 150 kDa, about 100kDa, or about 80 kDa; about 80 kDa to about 25,000 kDa, about 24,000kDa, about 22,000 kDa, about 20,000 kDa, about 18,000 kDa, about 16,000kDa, about 14,000 kDa, about 12,000 kDa, about 10,000 kDa, about 8,000kDa, about 6,000 kDa, about 4,000 kDa, about 2,000 kDa, about 1,800 kDa,about 1,600 kDa, about 1,400 kDa, about 1,200 kDa, about 1,000 kDa,about 950 kDa, about 900 kDa, about 850 kDa, about 800 kDa, about 750kDa, about 700 kDa, about 650 kDa, about 600 kDa, about 550 kDa, about500 kDa, about 450 kDa, about 400 kDa, about 350 kDa, about 300 kDa,about 250 kDa, about 200 kDa, about 150 kDa, or about 100 kDa; about 100kDa to about 25,000 kDa, about 24,000 kDa, about 22,000 kDa, about20,000 kDa, about 18,000 kDa, about 16,000 kDa, about 14,000 kDa, about12,000 kDa, about 10,000 kDa, about 8,000 kDa, about 6,000 kDa, about4,000 kDa, about 2,000 kDa, about 1,800 kDa, about 1,600 kDa, about1,400 kDa, about 1,200 kDa, about 1,000 kDa, about 950 kDa, about 900kDa, about 850 kDa, about 800 kDa, about 750 kDa, about 700 kDa, about650 kDa, about 600 kDa, about 550 kDa, about 500 kDa, about 450 kDa,about 400 kDa, about 350 kDa, about 300 kDa, about 250 kDa, about 200kDa, or about 150 kDa; about 150 kDa to about 25,000 kDa, about 24,000kDa, about 22,000 kDa, about 20,000 kDa, about 18,000 kDa, about 16,000kDa, about 14,000 kDa, about 12,000 kDa, about 10,000 kDa, about 8,000kDa, about 6,000 kDa, about 4,000 kDa, about 2,000 kDa, about 1,800 kDa,about 1,600 kDa, about 1,400 kDa, about 1,200 kDa, about 1,000 kDa,about 950 kDa, about 900 kDa, about 850 kDa, about 800 kDa, about 750kDa, about 700 kDa, about 650 kDa, about 600 kDa, about 550 kDa, about500 kDa, about 450 kDa, about 400 kDa, about 350 kDa, about 300 kDa,about 250 kDa, or about 200 kDa; about 200 kDa to about 25,000 kDa,about 24,000 kDa, about 22,000 kDa, about 20,000 kDa, about 18,000 kDa,about 16,000 kDa, about 14,000 kDa, about 12,000 kDa, about 10,000 kDa,about 8,000 kDa, about 6,000 kDa, about 4,000 kDa, about 2,000 kDa,about 1,800 kDa, about 1,600 kDa, about 1,400 kDa, about 1,200 kDa,about 1,000 kDa, about 950 kDa, about 900 kDa, about 850 kDa, about 800kDa, about 750 kDa, about 700 kDa, about 650 kDa, about 600 kDa, about550 kDa, about 500 kDa, about 450 kDa, about 400 kDa, about 350 kDa,about 300 kDa, or about 250 kDa; about 250 kDa to about 25,000 kDa,about 24,000 kDa, about 22,000 kDa, about 20,000 kDa, about 18,000 kDa,about 16,000 kDa, about 14,000 kDa, about 12,000 kDa, about 10,000 kDa,about 8,000 kDa, about 6,000 kDa, about 4,000 kDa, about 2,000 kDa,about 1,800 kDa, about 1,600 kDa, about 1,400 kDa, about 1,200 kDa,about 1,000 kDa, about 950 kDa, about 900 kDa, about 850 kDa, about 800kDa, about 750 kDa, about 700 kDa, about 650 kDa, about 600 kDa, about550 kDa, about 500 kDa, about 450 kDa, about 400 kDa, about 350 kDa, orabout 300 kDa; about 300 kDa to about 25,000 kDa, about 24,000 kDa,about 22,000 kDa, about 20,000 kDa, about 18,000 kDa, about 16,000 kDa,about 14,000 kDa, about 12,000 kDa, about 10,000 kDa, about 8,000 kDa,about 6,000 kDa, about 4,000 kDa, about 2,000 kDa, about 1,800 kDa,about 1,600 kDa, about 1,400 kDa, about 1,200 kDa, about 1,000 kDa,about 950 kDa, about 900 kDa, about 850 kDa, about 800 kDa, about 750kDa, about 700 kDa, about 650 kDa, about 600 kDa, about 550 kDa, about500 kDa, about 450 kDa, about 400 kDa, or about 350 kDa; about 350 kDato about 25,000 kDa, about 24,000 kDa, about 22,000 kDa, about 20,000kDa, about 18,000 kDa, about 16,000 kDa, about 14,000 kDa, about 12,000kDa, about 10,000 kDa, about 8,000 kDa, about 6,000 kDa, about 4,000kDa, about 2,000 kDa, about 1,800 kDa, about 1,600 kDa, about 1,400 kDa,about 1,200 kDa, about 1,000 kDa, about 950 kDa, about 900 kDa, about850 kDa, about 800 kDa, about 750 kDa, about 700 kDa, about 650 kDa,about 600 kDa, about 550 kDa, about 500 kDa, about 450 kDa, or about 400kDa; about 400 kDa to about 25,000 kDa, about 24,000 kDa, about 22,000kDa, about 20,000 kDa, about 18,000 kDa, about 16,000 kDa, about 14,000kDa, about 12,000 kDa, about 10,000 kDa, about 8,000 kDa, about 6,000kDa, about 4,000 kDa, about 2,000 kDa, about 1,800 kDa, about 1,600 kDa,about 1,400 kDa, about 1,200 kDa, about 1,000 kDa, about 950 kDa, about900 kDa, about 850 kDa, about 800 kDa, about 750 kDa, about 700 kDa,about 650 kDa, about 600 kDa, about 550 kDa, about 500 kDa, or about 450kDa; about 450 kDa to about 25,000 kDa, about 24,000 kDa, about 22,000kDa, about 20,000 kDa, about 18,000 kDa, about 16,000 kDa, about 14,000kDa, about 12,000 kDa, about 10,000 kDa, about 8,000 kDa, about 6,000kDa, about 4,000 kDa, about 2,000 kDa, about 1,800 kDa, about 1,600 kDa,about 1,400 kDa, about 1,200 kDa, about 1,000 kDa, about 950 kDa, about900 kDa, about 850 kDa, about 800 kDa, about 750 kDa, about 700 kDa,about 650 kDa, about 600 kDa, about 550 kDa, or about 500 kDa; about 500kDa to about 25,000 kDa, about 24,000 kDa, about 22,000 kDa, about20,000 kDa, about 18,000 kDa, about 16,000 kDa, about 14,000 kDa, about12,000 kDa, about 10,000 kDa, about 8,000 kDa, about 6,000 kDa, about4,000 kDa, about 2,000 kDa, about 1,800 kDa, about 1,600 kDa, about1,400 kDa, about 1,200 kDa, about 1,000 kDa, about 950 kDa, about 900kDa, about 850 kDa, about 800 kDa, about 750 kDa, about 700 kDa, about650 kDa, about 600 kDa, or about 550 kDa; about 550 kDa to about 25,000kDa, about 24,000 kDa, about 22,000 kDa, about 20,000 kDa, about 18,000kDa, about 16,000 kDa, about 14,000 kDa, about 12,000 kDa, about 10,000kDa, about 8,000 kDa, about 6,000 kDa, about 4,000 kDa, about 2,000 kDa,about 1,800 kDa, about 1,600 kDa, about 1,400 kDa, about 1,200 kDa,about 1,000 kDa, about 950 kDa, about 900 kDa, about 850 kDa, about 800kDa, about 750 kDa, about 700 kDa, about 650 kDa, or about 600 kDa;about 600 kDa to about 25,000 kDa, about 24,000 kDa, about 22,000 kDa,about 20,000 kDa, about 18,000 kDa, about 16,000 kDa, about 14,000 kDa,about 12,000 kDa, about 10,000 kDa, about 8,000 kDa, about 6,000 kDa,about 4,000 kDa, about 2,000 kDa, about 1,800 kDa, about 1,600 kDa,about 1,400 kDa, about 1,200 kDa, about 1,000 kDa, about 950 kDa, about900 kDa, about 850 kDa, about 800 kDa, about 750 kDa, about 700 kDa, orabout 650 kDa; about 650 kDa to about 25,000 kDa, about 24,000 kDa,about 22,000 kDa, about 20,000 kDa, about 18,000 kDa, about 16,000 kDa,about 14,000 kDa, about 12,000 kDa, about 10,000 kDa, about 8,000 kDa,about 6,000 kDa, about 4,000 kDa, about 2,000 kDa, about 1,800 kDa,about 1,600 kDa, about 1,400 kDa, about 1,200 kDa, about 1,000 kDa,about 950 kDa, about 900 kDa, about 850 kDa, about 800 kDa, about 750kDa, or about 700 kDa; about 700 kDa to about 25,000 kDa, about 24,000kDa, about 22,000 kDa, about 20,000 kDa, about 18,000 kDa, about 16,000kDa, about 14,000 kDa, about 12,000 kDa, about 10,000 kDa, about 8,000kDa, about 6,000 kDa, about 4,000 kDa, about 2,000 kDa, about 1,800 kDa,about 1,600 kDa, about 1,400 kDa, about 1,200 kDa, about 1,000 kDa,about 950 kDa, about 900 kDa, about 850 kDa, about 800 kDa, or about 750kDa; about 750 kDa to about 25,000 kDa, about 24,000 kDa, about 22,000kDa, about 20,000 kDa, about 18,000 kDa, about 16,000 kDa, about 14,000kDa, about 12,000 kDa, about 10,000 kDa, about 8,000 kDa, about 6,000kDa, about 4,000 kDa, about 2,000 kDa, about 1,800 kDa, about 1,600 kDa,about 1,400 kDa, about 1,200 kDa, about 1,000 kDa, about 950 kDa, about900 kDa, about 850 kDa, or about 800 kDa; about 800 kDa to about 25,000kDa, about 24,000 kDa, about 22,000 kDa, about 20,000 kDa, about 18,000kDa, about 16,000 kDa, about 14,000 kDa, about 12,000 kDa, about 10,000kDa, about 8,000 kDa, about 6,000 kDa, about 4,000 kDa, about 2,000 kDa,about 1,800 kDa, about 1,600 kDa, about 1,400 kDa, about 1,200 kDa,about 1,000 kDa, about 950 kDa, about 900 kDa, or about 850 kDa; about850 kDa to about 25,000 kDa, about 24,000 kDa, about 22,000 kDa, about20,000 kDa, about 18,000 kDa, about 16,000 kDa, about 14,000 kDa, about12,000 kDa, about 10,000 kDa, about 8,000 kDa, about 6,000 kDa, about4,000 kDa, about 2,000 kDa, about 1,800 kDa, about 1,600 kDa, about1,400 kDa, about 1,200 kDa, about 1,000 kDa, about 950 kDa, or about 900kDa; about 900 kDa to about 25,000 kDa, about 24,000 kDa, about 22,000kDa, about 20,000 kDa, about 18,000 kDa, about 16,000 kDa, about 14,000kDa, about 12,000 kDa, about 10,000 kDa, about 8,000 kDa, about 6,000kDa, about 4,000 kDa, about 2,000 kDa, about 1,800 kDa, about 1,600 kDa,about 1,400 kDa, about 1,200 kDa, about 1,000 kDa, or about 950 kDa;about 950 kDa to about 25,000 kDa, about 24,000 kDa, about 22,000 kDa,about 20,000 kDa, about 18,000 kDa, about 16,000 kDa, about 14,000 kDa,about 12,000 kDa, about 10,000 kDa, about 8,000 kDa, about 6,000 kDa,about 4,000 kDa, about 2,000 kDa, about 1,800 kDa, about 1,600 kDa,about 1,400 kDa, about 1,200 kDa, or about 1,000 kDa; about 1,000 kDa toabout 25,000 kDa, about 24,000 kDa, about 22,000 kDa, about 20,000 kDa,about 18,000 kDa, about 16,000 kDa, about 14,000 kDa, about 12,000 kDa,about 10,000 kDa, about 8,000 kDa, about 6,000 kDa, about 4,000 kDa,about 2,000 kDa, about 1,800 kDa, about 1,600 kDa, about 1,400 kDa, orabout 1,200 kDa; about 1,200 kDa to about 25,000 kDa, about 24,000 kDa,about 22,000 kDa, about 20,000 kDa, about 18,000 kDa, about 16,000 kDa,about 14,000 kDa, about 12,000 kDa, about 10,000 kDa, about 8,000 kDa,about 6,000 kDa, about 4,000 kDa, about 2,000 kDa, about 1,800 kDa,about 1,600 kDa, or about 1,400 kDa; about 1,400 kDa to about 25,000kDa, about 24,000 kDa, about 22,000 kDa, about 20,000 kDa, about 18,000kDa, about 16,000 kDa, about 14,000 kDa, about 12,000 kDa, about 10,000kDa, about 8,000 kDa, about 6,000 kDa, about 4,000 kDa, about 2,000 kDa,about 1,800 kDa, or about 1,600 kDa; about 1,600 kDa to about 25,000kDa, about 24,000 kDa, about 22,000 kDa, about 20,000 kDa, about 18,000kDa, about 16,000 kDa, about 14,000 kDa, about 12,000 kDa, about 10,000kDa, about 8,000 kDa, about 6,000 kDa, about 4,000 kDa, about 2,000 kDa,or about 1,800 kDa; about 1,800 kDa to about 25,000 kDa, about 24,000kDa, about 22,000 kDa, about 20,000 kDa, about 18,000 kDa, about 16,000kDa, about 14,000 kDa, about 12,000 kDa, about 10,000 kDa, about 8,000kDa, about 6,000 kDa, about 4,000 kDa, or about 2,000 kDa; about 2,000kDa to about 25,000 kDa, about 24,000 kDa, about 22,000 kDa, about20,000 kDa, about 18,000 kDa, about 16,000 kDa, about 14,000 kDa, about12,000 kDa, about 10,000 kDa, about 8,000 kDa, about 6,000 kDa, or about4,000 kDa; about 4,000 kDa to about 25,000 kDa, about 24,000 kDa, about22,000 kDa, about 20,000 kDa, about 18,000 kDa, about 16,000 kDa, about14,000 kDa, about 12,000 kDa, about 10,000 kDa, about 8,000 kDa, orabout 6,000 kDa; about 6,000 kDa to about 25,000 kDa, about 24,000 kDa,about 22,000 kDa, about 20,000 kDa, about 18,000 kDa, about 16,000 kDa,about 14,000 kDa, about 12,000 kDa, about 10,000 kDa, or about 8,000kDa; about 8,000 kDa to about 25,000 kDa, about 24,000 kDa, about 22,000kDa, about 20,000 kDa, about 18,000 kDa, about 16,000 kDa, about 14,000kDa, about 12,000 kDa, or about 10,000 kDa; about 10,000 kDa to about25,000 kDa, about 24,000 kDa, about 22,000 kDa, about 20,000 kDa, about18,000 kDa, about 16,000 kDa, about 14,000 kDa, or about 12,000 kDa;about 12,000 kDa to about 25,000 kDa, about 24,000 kDa, about 22,000kDa, about 20,000 kDa, about 18,000 kDa, about 16,000 kDa, or about14,000 kDa; about 14,000 kDa to about 25,000 kDa, about 24,000 kDa,about 22,000 kDa, about 20,000 kDa, about 18,000 kDa, or about 16,000kDa; about 16,000 kDa to about 25,000 kDa, about 24,000 kDa, about22,000 kDa, about 20,000 kDa, or about 18,000 kDa; about 18,000 kDa toabout 25,000 kDa, about 24,000 kDa, about 22,000 kDa, or about 20,000kDa; about 20,000 kDa to about 25,000 kDa, about 24,000 kDa, or about22,000 kDa; about 22,000 kDa to about 25,000 kDa or about 24,000 kDa; orabout 23,000 kDa to about 25,000 kDa. In some embodiments, the TFF unitincludes one or more tangential filters made of, e.g., cellulose (e.g.,regenerated cellulose) or polyethersulfone. Non-limiting examples of oneor more tangential filters that can be used in the TFF unit arecommercially available from Millipore (e.g., Ultracel® and Biomax®).Additional commercial sources of one or more tangential filters that canbe used in the TFF unit are known in the art.

The conduit can be simple tubing, e.g., biocompatible tubing.Non-limiting examples of useful tubing include silicone rubber,polyurethane, polydioxanone (PDO), polyhydroxyalkanoate,polyhydroxybutyrate, poly(glycerol sebacate), polyglycolide,polylactide, polycaprolactone, or polyanhydride, or copolymers orderivatives including these and/or other polymers. Alternatively or inaddition, any of the conduits described herein can include polyvinylchloride. A conduit can be, e.g., weldable transfer tubing.

In some embodiments, the internal diameter of the conduit can be about 5mm to about 50 mm, about 48 mm, about 46 mm, about 44 mm, about 42 mm,about 40 mm, about 38 mm, about 36 mm, about 34 mm, about 32 mm, about30 mm, about 28 mm, about 26 mm, about 24 mm, about 22 mm, about 20 mm,about 18 mm, about 16 mm, about 14 mm, about 12 mm, about 10 mm, about 8mm, or about 6 mm; about 6 mm to about 50 mm, about 48 mm, about 46 mm,about 44 mm, about 42 mm, about 40 mm, about 38 mm, about 36 mm, about34 mm, about 32 mm, about 30 mm, about 28 mm, about 26 mm, about 24 mm,about 22 mm, about 20 mm, about 18 mm, about 16 mm, about 14 mm, about12 mm, about 10 mm, or about 8 mm; about 8 mm to about 50 mm, about 48mm, about 46 mm, about 44 mm, about 42 mm, about 40 mm, about 38 mm,about 36 mm, about 34 mm, about 32 mm, about 30 mm, about 28 mm, about26 mm, about 24 mm, about 22 mm, about 20 mm, about 18 mm, about 16 mm,about 14 mm, about 12 mm, or about 10 mm; about 10 mm to about 50 mm,about 48 mm, about 46 mm, about 44 mm, about 42 mm, about 40 mm, about38 mm, about 36 mm, about 34 mm, about 32 mm, about 30 mm, about 28 mm,about 26 mm, about 24 mm, about 22 mm, about 20 mm, about 18 mm, about16 mm, about 14 mm, or about 12 mm; about 12 mm to about 50 mm, about 48mm, about 46 mm, about 44 mm, about 42 mm, about 40 mm, about 38 mm,about 36 mm, about 34 mm, about 32 mm, about 30 mm, about 28 mm, about26 mm, about 24 mm, about 22 mm, about 20 mm, about 18 mm, about 16 mm,or about 14 mm; about 14 mm to about 50 mm, about 48 mm, about 46 mm,about 44 mm, about 42 mm, about 40 mm, about 38 mm, about 36 mm, about34 mm, about 32 mm, about 30 mm, about 28 mm, about 26 mm, about 24 mm,about 22 mm, about 20 mm, about 18 mm, or about 16 mm; about 16 mm toabout 50 mm, about 48 mm, about 46 mm, about 44 mm, about 42 mm, about40 mm, about 38 mm, about 36 mm, about 34 mm, about 32 mm, about 30 mm,about 28 mm, about 26 mm, about 24 mm, about 22 mm, about 20 mm, orabout 18 mm; about 18 mm to about 50 mm, about 48 mm, about 46 mm, about44 mm, about 42 mm, about 40 mm, about 38 mm, about 36 mm, about 34 mm,about 32 mm, about 30 mm, about 28 mm, about 26 mm, about 24 mm, about22 mm, or about 20 mm; about 20 mm to about 50 mm, about 48 mm, about 46mm, about 44 mm, about 42 mm, about 40 mm, about 38 mm, about 36 mm,about 34 mm, about 32 mm, about 30 mm, about 28 mm, about 26 mm, about24 mm, or about 22 mm; about 22 mm to about 50 mm, about 48 mm, about 46mm, about 44 mm, about 42 mm, about 40 mm, about 38 mm, about 36 mm,about 34 mm, about 32 mm, about 30 mm, about 28 mm, about 26 mm, orabout 24 mm; about 24 mm to about 50 mm, about 48 mm, about 46 mm, about44 mm, about 42 mm, about 40 mm, about 38 mm, about 36 mm, about 34 mm,about 32 mm, about 30 mm, about 28 mm, or about 26 mm; about 26 mm toabout 50 mm, about 48 mm, about 46 mm, about 44 mm, about 42 mm, about40 mm, about 38 mm, about 36 mm, about 34 mm, about 32 mm, about 30 mm,or about 28 mm; about 28 mm to about 50 mm, about 48 mm, about 46 mm,about 44 mm, about 42 mm, about 40 mm, about 38 mm, about 36 mm, about34 mm, about 32 mm, or about 30 mm; about 30 mm to about 50 mm, about 48mm, about 46 mm, about 44 mm, about 42 mm, about 40 mm, about 38 mm,about 36 mm, about 34 mm, or about 32 mm; about 32 mm to about 50 mm,about 48 mm, about 46 mm, about 44 mm, about 42 mm, about 40 mm, about38 mm, about 36 mm, or about 34 mm; about 34 mm to about 50 mm, about 48mm, about 46 mm, about 44 mm, about 42 mm, about 40 mm, about 38 mm, orabout 36 mm; about 36 mm to about 50 mm, about 48 mm, about 46 mm, about44 mm, about 42 mm, about 40 mm, to about 38 mm; about 38 mm to about 50mm, about 48 mm, about 46 mm, about 44 mm, about 42 mm, or about 40 mm;about 40 mm to about 50 mm, about 48 mm, about 46 mm, about 44 mm, orabout 42 mm; about 42 mm to about 50 mm, about 48 mm, about 46 mm, orabout 44 mm; about 44 mm to about 50 mm, about 48 mm, or about 46 mm;about 46 mm to about 50 mm or about 48 mm; or about 48 mm to about 50mm.

In some embodiments, the conduit has a total length of about 1.0 cm toabout 75 cm, about 70 cm, about 65 cm, about 60 cm, about 55 cm, about50 cm, about 45 cm, about 40 cm, about 35 cm, about 30 cm, about 25 cm,about 20 cm, about 18 cm, about 16 cm, about 14 cm, about 12 cm, about10 cm, about 8.0 cm, about 6.0 cm, about 4.0 cm, or about 2.0 cm; about2.0 cm to about 75 cm, about 70 cm, about 65 cm, about 60 cm, about 55cm, about 50 cm, about 45 cm, about 40 cm, about 35 cm, about 30 cm,about 25 cm, about 20 cm, about 18 cm, about 16 cm, about 14 cm, about12 cm, about 10 cm, about 8.0 cm, about 6.0 cm, or about 4.0 cm; about4.0 cm to about 75 cm, about 70 cm, about 65 cm, about 60 cm, about 55cm, about 50 cm, about 45 cm, about 40 cm, about 35 cm, about 30 cm,about 25 cm, about 20 cm, about 18 cm, about 16 cm, about 14 cm, about12 cm, about 10 cm, about 8.0 cm, or about 6.0 cm; about 6.0 cm to about75 cm, about 70 cm, about 65 cm, about 60 cm, about 55 cm, about 50 cm,about 45 cm, about 40 cm, about 35 cm, about 30 cm, about 25 cm, about20 cm, about 18 cm, about 16 cm, about 14 cm, about 12 cm, about 10 cm,or about 8.0 cm; about 8.0 cm to about 75 cm, about 70 cm, about 65 cm,about 60 cm, about 55 cm, about 50 cm, about 45 cm, about 40 cm, about35 cm, about 30 cm, about 25 cm, about 20 cm, about 18 cm, about 16 cm,about 14 cm, about 12 cm, or about 10 cm; about 10 cm to about 75 cm,about 70 cm, about 65 cm, about 60 cm, about 55 cm, about 50 cm, about45 cm, about 40 cm, about 35 cm, about 30 cm, about 25 cm, about 20 cm,about 18 cm, about 16 cm, about 14 cm, or about 12 cm; about 12 cm toabout 75 cm, about 70 cm, about 65 cm, about 60 cm, about 55 cm, about50 cm, about 45 cm, about 40 cm, about 35 cm, about 30 cm, about 25 cm,about 20 cm, about 18 cm, about 16 cm, or about 14 cm; about 14 cm toabout 75 cm, about 70 cm, about 65 cm, about 60 cm, about 55 cm, about50 cm, about 45 cm, about 40 cm, about 35 cm, about 30 cm, about 25 cm,about 20 cm, about 18 cm, or about 16 cm; about 16 cm to about 75 cm,about 70 cm, about 65 cm, about 60 cm, about 55 cm, about 50 cm, about45 cm, about 40 cm, about 35 cm, about 30 cm, about 25 cm, about 20 cm,or about 18 cm; about 18 cm to about 75 cm, about 70 cm, about 65 cm,about 60 cm, about 55 cm, about 50 cm, about 45 cm, about 40 cm, about35 cm, about 30 cm, about 25 cm, or about 20 cm; about 20 cm to about 75cm, about 70 cm, about 65 cm, about 60 cm, about 55 cm, about 50 cm,about 45 cm, about 40 cm, about 35 cm, about 30 cm, or about 25 cm;about 25 cm to about 75 cm, about 70 cm, about 65 cm, about 60 cm, about55 cm, about 50 cm, about 45 cm, about 40 cm, about 35 cm, or about 30cm; about 30 cm to about 75 cm, about 70 cm, about 65 cm, about 60 cm,about 55 cm, about 50 cm, about 45 cm, about 40 cm, or about 35 cm;about 35 cm to about 75 cm, about 70 cm, about 65 cm, about 60 cm, about55 cm, about 50 cm, about 45 cm, or about 40 cm; about 40 cm to about 75cm, about 70 cm, about 65 cm, about 60 cm, about 55 cm, about 50 cm, orabout 45 cm; about 45 cm to about 75 cm, about 70 cm, about 65 cm, about60 cm, about 55 cm, or about 50 cm; about 50 cm to about 75 cm, about 70cm, about 65 cm, about 60 cm, or about 55 cm; about 55 cm to about 75cm, about 70 cm, about 65 cm, or about 60 cm; about 60 cm to about 75cm, about 70 cm, or about 65 cm; about 65 cm to about 75 cm or about 70cm; or about 70 cm to about 75 cm.

Additional examples of conduits and properties of conduits that can beused in the circuit system are well-known by those in the art.

In some examples, the circuit system includes one, two, three, four, orfive ports. In some examples, the at least one port can be a valve. Inother examples, the at least one port can be an injection port or canhave a ribbed threading. The at least one port can be any type of portcommonly known in the art.

In some examples, the circuit system can further include a pump (e.g., alow-turbulence pump, e.g., a peristaltic pump) disposed in the conduit.

In some examples, step (b) includes continuously flowing (e.g.,unidirectionally or bidirectionally flowing) the fluid including therecombinant therapeutic protein into the circuit system at a rate ofbetween about 0.05 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, about 7.0mL/minute, about 6.5 mL/minute, about 6.0 mL/minute, about 5.5mL/minute, about 5.0 mL/minute, about 4.5 mL/minute, about 4.0mL/minute, about 3.5 mL/minute, about 3.0 mL/minute, about 2.5mL/minute, about 2.0 mL/minute, about 1.5 mL/minute, about 1.0mL/minute, about 0.5 mL/minute, or about 0.1 mL/minute; about 0.1mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, about 6.5mL/minute, about 6.0 mL/minute, about 5.5 mL/minute, about 5.0mL/minute, about 4.5 mL/minute, about 4.0 mL/minute, about 3.5mL/minute, about 3.0 mL/minute, about 2.5 mL/minute, about 2.0mL/minute, about 1.5 mL/minute, about 1.0 mL/minute, or about 0.5mL/minute; about 0.5 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, about 7.0mL/minute, about 6.5 mL/minute, about 6.0 mL/minute, about 5.5mL/minute, about 5.0 mL/minute, about 4.5 mL/minute, about 4.0mL/minute, about 3.5 mL/minute, about 3.0 mL/minute, about 2.5mL/minute, about 2.0 mL/minute, about 1.5 mL/minute, or about 1.0mL/minute; about 1.0 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, about 7.0mL/minute, about 6.5 mL/minute, about 6.0 mL/minute, about 5.5mL/minute, about 5.0 mL/minute, about 4.5 mL/minute, about 4.0mL/minute, about 3.5 mL/minute, about 3.0 mL/minute, about 2.5mL/minute, about 2.0 mL/minute, or about 1.5 mL/minute; about 1.5mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, about 6.5mL/minute, about 6.0 mL/minute, about 5.5 mL/minute, about 5.0mL/minute, about 4.5 mL/minute, about 4.0 mL/minute, about 3.5mL/minute, about 3.0 mL/minute, about 2.5 mL/minute, or about 2.0mL/minute; about 2.0 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, about 7.0mL/minute, about 6.5 mL/minute, about 6.0 mL/minute, about 5.5mL/minute, about 5.0 mL/minute, about 4.5 mL/minute, about 4.0mL/minute, about 3.5 mL/minute, about 3.0 mL/minute, or about 2.5mL/minute; about 2.5 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, about 7.0mL/minute, about 6.5 mL/minute, about 6.0 mL/minute, about 5.5mL/minute, about 5.0 mL/minute, about 4.5 mL/minute, about 4.0mL/minute, about 3.5 mL/minute, or about 3.0 mL/minute; about 3.0mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, about 6.5mL/minute, about 6.0 mL/minute, about 5.5 mL/minute, about 5.0mL/minute, about 4.5 mL/minute, about 4.0 mL/minute, or about 3.5mL/minute; about 3.5 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, about 7.0mL/minute, about 6.5 mL/minute, about 6.0 mL/minute, about 5.5mL/minute, about 5.0 mL/minute, about 4.5 mL/minute, or about 4.0mL/minute; about 4.0 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, about 7.0mL/minute, about 6.5 mL/minute, about 6.0 mL/minute, about 5.5mL/minute, about 5.0 mL/minute, or about 4.5 mL/minute; about 4.5mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, about 6.5mL/minute, about 6.0 mL/minute, about 5.5 mL/minute, or about 5.0mL/minute; about 5.0 mL/minute to about 25 mL/minute, about 24mL/minute, about 22 mL/minute, about 20 mL/minute, about 18 mL/minute,about 16 mL/minute, about 14 mL/minute, about 12 mL/minute, about 10mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, about 7.0mL/minute, about 6.5 mL/minute, about 6.0 mL/minute, or about 5.5mL/minute; about 5.5 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, about 7.0mL/minute, about 6.5 mL/minute, or about 6.0 mL/minute; about 6.0mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, or about 6.5mL/minute; about 6.5 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, or about 7.0mL/minute; about 7.0 mL/minute to about 25 mL/minute, about 24mL/minute, about 22 mL/minute, about 20 mL/minute, about 18 mL/minute,about 16 mL/minute, about 14 mL/minute, about 12 mL/minute, about 10mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, or about 7.5 mL/minute; about 7.5mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, or about8.0 mL/minute; about 8.0 mL/minute to about 100 L/minute, about 90L/minute, about 80 L/minute, about 70 L/minute, about 60 L/minute, about50 L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute,about 10 L/minute, about 5 L/minute, about 1 L/minute, about 950mL/minute, about 900 mL/minute, about 850 mL/minute, about 800mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute, about 24 mL/minute, about 22 mL/minute, about 20mL/minute, about 18 mL/minute, about 16 mL/minute, about 14 mL/minute,about 12 mL/minute, about 10 mL/minute, about 9.5 mL/minute, about 9.0mL/minute, or about 8.5 mL/minute; about 8.5 mL/minute to about 25mL/minute, about 24 mL/minute, about 22 mL/minute, about 20 mL/minute,about 18 mL/minute, about 16 mL/minute, about 14 mL/minute, about 12mL/minute, about 10 mL/minute, about 9.5 mL/minute, or about 9.0mL/minute; about 9.0 mL/minute to about 25 mL/minute, about 24mL/minute, about 22 mL/minute, about 20 mL/minute, about 18 mL/minute,about 16 mL/minute, about 14 mL/minute, about 12 mL/minute, about 10mL/minute, or about 9.5 mL/minute; about 9.5 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,about 800 mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute, about 24 mL/minute, about 22 mL/minute, about 20mL/minute, about 18 mL/minute, about 16 mL/minute, about 14 mL/minute,about 12 mL/minute, or about 10 mL/minute; about 10 mL/minute to about100 L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute,about 60 L/minute, about 50 L/minute, about 40 L/minute, about 30L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, or about 12 mL/minute; about 12 mL/minuteto about 100 L/minute, about 90 L/minute, about 80 L/minute, about 70L/minute, about 60 L/minute, about 50 L/minute, about 40 L/minute, about30 L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute,about 1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, or about 14 mL/minute; about 14 mL/minute to about 25mL/minute, about 24 mL/minute, about 22 mL/minute, about 20 mL/minute,about 18 mL/minute, or about 16 mL/minute; about 16 mL/minute to about100 L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute,about 60 L/minute, about 50 L/minute, about 40 L/minute, about 30L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, or about 18 mL/minute; about 18mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, or about 20 mL/minute; about 20 mL/minute to about100 L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute,about 60 L/minute, about 50 L/minute, about 40 L/minute, about 30L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,or about 22 mL/minute; about 22 mL/minute to about 100 L/minute, about90 L/minute, about 80 L/minute, about 70 L/minute, about 60 L/minute,about 50 L/minute, about 40 L/minute, about 30 L/minute, about 20L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about950 mL/minute, about 900 mL/minute, about 850 mL/minute, about 800mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute or about 24 mL/minute; about 23 mL/minute to about100 L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute,about 60 L/minute, about 50 L/minute, about 40 L/minute, about 30L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, or about 25 mL/minute; about 25 mL/minuteto about 100 L/minute, about 90 L/minute, about 80 L/minute, about 70L/minute, about 60 L/minute, about 50 L/minute, about 40 L/minute, about30 L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute,about 1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, or about 50 mL/minute; about 50 mL to about 100 L/minute,about 90 L/minute, about 80 L/minute, about 70 L/minute, about 60L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute, about20 L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute,about 950 mL/minute, about 900 mL/minute, about 850 mL/minute, about 800mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, or about 100 mL/minute; about 100mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, or about 150 mL/minute; about 150mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, or about 200 mL/minute; about 200 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,about 800 mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, or about 250 mL/minute; about 250mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, or about 300 mL/minute; about 300mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, or about 350 mL/minute; about 350 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,about 800 mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, or about 400 mL/minute; about 400mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, or about 450 mL/minute; about 450mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, or about 500 mL/minute; about 500 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,about 800 mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, or about 550 mL/minute; about 550 mL toabout 100 L/minute, about 90 L/minute, about 80 L/minute, about 70L/minute, about 60 L/minute, about 50 L/minute, about 40 L/minute, about30 L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute,about 1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, or about 600 mL/minute; about 600 mL toabout 100 L/minute, about 90 L/minute, about 80 L/minute, about 70L/minute, about 60 L/minute, about 50 L/minute, about 40 L/minute, about30 L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute,about 1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, or about 650 mL/minute; about 650 mL to about 100 L/minute,about 90 L/minute, about 80 L/minute, about 70 L/minute, about 60L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute, about20 L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute,about 950 mL/minute, about 900 mL/minute, about 850 mL/minute, about 800mL/minute, about 750 mL/minute, or about 700 mL/minute; about 700mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, or about 750 mL/minute; about750 mL/minute to about 100 L/minute, about 90 L/minute, about 80L/minute, about 70 L/minute, about 60 L/minute, about 50 L/minute, about40 L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute,about 5 L/minute, about 1 L/minute, about 950 mL/minute, about 900mL/minute, about 850 mL/minute, or about 800 mL/minute; about 800mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,or about 850 mL/minute; about 850 mL/minute to about 100 L/minute, about90 L/minute, about 80 L/minute, about 70 L/minute, about 60 L/minute,about 50 L/minute, about 40 L/minute, about 30 L/minute, about 20L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about950 mL/minute, or about 900 mL/minute; about 900 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, or about 950 mL/minute; about 950 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, or about 1L/minute; about 1 L/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, or about 5 L/minute; about 5 L/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, or about 10 L/minute; about 10 L/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute, orabout 20 L/minute; about 20 L/minute to about 100 L/minute, about 90L/minute, about 80 L/minute, about 70 L/minute, about 60 L/minute, about50 L/minute, about 40 L/minute, or about 30 L/minute; about 30 L/minuteto about 100 L/minute, about 90 L/minute, about 80 L/minute, about 70L/minute, about 60 L/minute, about 50 L/minute, or about 40 L/minute;about 40 L/minute to about 100 L/minute, about 90 L/minute, about 80L/minute, about 70 L/minute, about 60 L/minute, or about 50 L/minute;about 50 L/minute to about 100 L/minute, about 90 L/minute, about 80L/minute, about 70 L/minute, or about 60 L/minute; about 60 L/minute toabout 100 L/minute, about 90 L/minute, about 80 L/minute, or about 70L/minute; about 70 L/minute to about 100 L/minute, about 90 L/minute, orabout 80 L/minute; about 80 L/minute to about 100 L/minute, or about 90L/minute; or about 90 L/minute to about 100 L/minute.

In some examples, step (b) includes continuously flowing (e.g.,unidirectionally or bidirectionally flowing) a fluid including about0.01 mg/mL to about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL,about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL, about 26mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL, about 18 mg/mL,about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, about 10 mg/mL, 9.5mg/mL, about 9.0 mg/mL, about 8.5 mg/mL, about 8.0 mg/mL, about 7.5mg/mL, about 7.0 mg/mL, about 6.5 mg/mL, about 6.0 mg/mL, about 5.5mg/mL, about 5.0 mg/mL, about 4.5 mg/mL, about 4.0 mg/mL, about 3.5mg/mL, about 3.0 mg/mL, about 2.5 mg/mL, about 2.0 mg/mL, about 1.5mg/mL, about 1.0 mg/mL, about 0.5 mg/mL, about 0.1 mg/mL, or about 0.05mg/mL; about 0.05 mg/mL to about 50 mg/mL, about 48 mg/mL, about 46mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL,about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL,about 18 mg/mL, about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, about 10mg/mL, 9.5 mg/mL, about 9.0 mg/mL, about 8.5 mg/mL, about 8.0 mg/mL,about 7.5 mg/mL, about 7.0 mg/mL, about 6.5 mg/mL, about 6.0 mg/mL,about 5.5 mg/mL, about 5.0 mg/mL, about 4.5 mg/mL, about 4.0 mg/mL,about 3.5 mg/mL, about 3.0 mg/mL, about 2.5 mg/mL, about 2.0 mg/mL,about 1.5 mg/mL, about 1.0 mg/mL, about 0.5 mg/mL, or about 0.1 mg/mL;about 0.1 mg/mL to about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL,about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL, about 18mg/mL, about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, about 10 mg/mL,9.5 mg/mL, about 9.0 mg/mL, about 8.5 mg/mL, about 8.0 mg/mL, about 7.5mg/mL, about 7.0 mg/mL, about 6.5 mg/mL, about 6.0 mg/mL, about 5.5mg/mL, about 5.0 mg/mL, about 4.5 mg/mL, about 4.0 mg/mL, about 3.5mg/mL, about 3.0 mg/mL, about 2.5 mg/mL, about 2.0 mg/mL, about 1.5mg/mL, about 1.0 mg/mL, or about 0.5 mg/mL; about 0.5 mg/mL to about 50mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL,about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL,about 22 mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14mg/mL, about 12 mg/mL, about 10 mg/mL, 9.5 mg/mL, about 9.0 mg/mL, about8.5 mg/mL, about 8.0 mg/mL, about 7.5 mg/mL, about 7.0 mg/mL, about 6.5mg/mL, about 6.0 mg/mL, about 5.5 mg/mL, about 5.0 mg/mL, about 4.5mg/mL, about 4.0 mg/mL, about 3.5 mg/mL, about 3.0 mg/mL, about 2.5mg/mL, about 2.0 mg/mL, about 1.5 mg/mL, or about 1.0 mg/mL; about 1.0mg/mL to about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL,about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL,about 24 mg/mL, about 22 mg/mL, about 20 mg/mL, about 18 mg/mL, about 16mg/mL, about 14 mg/mL, about 12 mg/mL, about 10 mg/mL, 9.5 mg/mL, about9.0 mg/mL, about 8.5 mg/mL, about 8.0 mg/mL, about 7.5 mg/mL, about 7.0mg/mL, about 6.5 mg/mL, about 6.0 mg/mL, about 5.5 mg/mL, about 5.0mg/mL, about 4.5 mg/mL, about 4.0 mg/mL, about 3.5 mg/mL, about 3.0mg/mL, about 2.5 mg/mL, about 2.0 mg/mL, or about 1.5 mg/mL; about 1.5mg/mL to about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL,about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL,about 24 mg/mL, about 22 mg/mL, about 20 mg/mL, about 18 mg/mL, about 16mg/mL, about 14 mg/mL, about 12 mg/mL, about 10 mg/mL, 9.5 mg/mL, about9.0 mg/mL, about 8.5 mg/mL, about 8.0 mg/mL, about 7.5 mg/mL, about 7.0mg/mL, about 6.5 mg/mL, about 6.0 mg/mL, about 5.5 mg/mL, about 5.0mg/mL, about 4.5 mg/mL, about 4.0 mg/mL, about 3.5 mg/mL, about 3.0mg/mL, about 2.5 mg/mL, or about 2.0 mg/mL; about 2.0 mg/mL to about 50mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL,about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL,about 22 mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14mg/mL, about 12 mg/mL, about 10 mg/mL, 9.5 mg/mL, about 9.0 mg/mL, about8.5 mg/mL, about 8.0 mg/mL, about 7.5 mg/mL, about 7.0 mg/mL, about 6.5mg/mL, about 6.0 mg/mL, about 5.5 mg/mL, about 5.0 mg/mL, about 4.5mg/mL, about 4.0 mg/mL, about 3.5 mg/mL, about 3.0 mg/mL, or about 2.5mg/mL; about 2.5 mg/mL to about 50 mg/mL, about 48 mg/mL, about 46mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL,about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL,about 18 mg/mL, about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, about 10mg/mL, 9.5 mg/mL, about 9.0 mg/mL, about 8.5 mg/mL, about 8.0 mg/mL,about 7.5 mg/mL, about 7.0 mg/mL, about 6.5 mg/mL, about 6.0 mg/mL,about 5.5 mg/mL, about 5.0 mg/mL, about 4.5 mg/mL, about 4.0 mg/mL,about 3.5 mg/mL, or about 3.0 mg/mL; about 3.0 mg/mL to about 50 mg/mL,about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL,about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14 mg/mL,about 12 mg/mL, about 10 mg/mL, 9.5 mg/mL, about 9.0 mg/mL, about 8.5mg/mL, about 8.0 mg/mL, about 7.5 mg/mL, about 7.0 mg/mL, about 6.5mg/mL, about 6.0 mg/mL, about 5.5 mg/mL, about 5.0 mg/mL, about 4.5mg/mL, about 4.0 mg/mL, or about 3.5 mg/mL; about 3.5 mg/mL to about 50mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL,about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL,about 22 mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14mg/mL, about 12 mg/mL, about 10 mg/mL, 9.5 mg/mL, about 9.0 mg/mL, about8.5 mg/mL, about 8.0 mg/mL, about 7.5 mg/mL, about 7.0 mg/mL, about 6.5mg/mL, about 6.0 mg/mL, about 5.5 mg/mL, about 5.0 mg/mL, about 4.5mg/mL, or about 4.0 mg/mL; about 4.0 mg/mL to about 50 mg/mL, about 48mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL,about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL,about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14 mg/mL, about 12mg/mL, about 10 mg/mL, 9.5 mg/mL, about 9.0 mg/mL, about 8.5 mg/mL,about 8.0 mg/mL, about 7.5 mg/mL, about 7.0 mg/mL, about 6.5 mg/mL,about 6.0 mg/mL, about 5.5 mg/mL, about 5.0 mg/mL, or about 4.5 mg/mL;about 4.5 mg/mL to about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL,about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL, about 18mg/mL, about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, about 10 mg/mL,9.5 mg/mL, about 9.0 mg/mL, about 8.5 mg/mL, about 8.0 mg/mL, about 7.5mg/mL, about 7.0 mg/mL, about 6.5 mg/mL, about 6.0 mg/mL, about 5.5mg/mL, or about 5.0 mg/mL; about 5.0 mg/mL to about 50 mg/mL, about 48mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL,about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL,about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14 mg/mL, about 12mg/mL, about 10 mg/mL, 9.5 mg/mL, about 9.0 mg/mL, about 8.5 mg/mL,about 8.0 mg/mL, about 7.5 mg/mL, about 7.0 mg/mL, about 6.5 mg/mL,about 6.0 mg/mL, or about 5.5 mg/mL; about 5.5 mg/mL to about 50 mg/mL,about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL,about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14 mg/mL,about 12 mg/mL, about 10 mg/mL, 9.5 mg/mL, about 9.0 mg/mL, about 8.5mg/mL, about 8.0 mg/mL, about 7.5 mg/mL, about 7.0 mg/mL, about 6.5mg/mL, or about 6.0 mg/mL; about 6.0 mg/mL to about 50 mg/mL, about 48mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL,about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL,about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14 mg/mL, about 12mg/mL, about 10 mg/mL, 9.5 mg/mL, about 9.0 mg/mL, about 8.5 mg/mL,about 8.0 mg/mL, about 7.5 mg/mL, about 7.0 mg/mL, or about 6.5 mg/mL;about 6.5 mg/mL to about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL,about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL, about 18mg/mL, about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, about 10 mg/mL,9.5 mg/mL, about 9.0 mg/mL, about 8.5 mg/mL, about 8.0 mg/mL, about 7.5mg/mL, or about 7.0 mg/mL; about 7.0 mg/mL to about 50 mg/mL, about 48mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL,about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL,about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14 mg/mL, about 12mg/mL, about 10 mg/mL, 9.5 mg/mL, about 9.0 mg/mL, about 8.5 mg/mL,about 8.0 mg/mL, or about 7.5 mg/mL; about 7.5 mg/mL to about 50 mg/mL,about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL,about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14 mg/mL,about 12 mg/mL, about 10 mg/mL, 9.5 mg/mL, about 9.0 mg/mL, about 8.5mg/mL, or about 8.0 mg/mL; about 8.0 mg/mL to about 50 mg/mL, about 48mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL,about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL,about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14 mg/mL, about 12mg/mL, about 10 mg/mL, 9.5 mg/mL, about 9.0 mg/mL, or about 8.5 mg/mL;about 8.5 mg/mL to about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL,about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL, about 18mg/mL, about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, about 10 mg/mL,9.5 mg/mL, or about 9.0 mg/mL; about 9.0 mg/mL to about 50 mg/mL, about48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL,about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14 mg/mL,about 12 mg/mL, about 10 mg/mL, or 9.5 mg/mL; about 9.5 mg/mL to about50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL,about 32 mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24mg/mL, about 22 mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL,about 14 mg/mL, about 12 mg/mL, or about 10 mg/mL; about 10 mg/mL toabout 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL,about 32 mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24mg/mL, about 22 mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL,about 14 mg/mL, or about 12 mg/mL; about 12 mg/mL to about 50 mg/mL,about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL,about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, or about 14mg/mL; about 14 mg/mL to about 50 mg/mL, about 48 mg/mL, about 46 mg/mL,about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL,about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL, about 18mg/mL, or about 16 mg/mL; about 16 mg/mL to about 50 mg/mL, about 48mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL,about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL,about 20 mg/mL, or about 18 mg/mL; about 18 mg/mL to about 50 mg/mL,about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL,about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22mg/mL, or about 20 mg/mL; about 20 mg/mL to about 50 mg/mL, about 48mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL,about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, or about 22mg/mL; about 22 mg/mL to about 50 mg/mL, about 48 mg/mL, about 46 mg/mL,about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL,about 26 mg/mL, or about 24 mg/mL; about 24 mg/mL to about 50 mg/mL,about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL,about 30 mg/mL, about 28 mg/mL, or about 26 mg/mL; about 26 mg/mL toabout 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL,about 32 mg/mL, about 30 mg/mL, or about 28 mg/mL; about 28 mg/mL toabout 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL,about 32 mg/mL, or about 30 mg/mL; about 30 mg/mL to about 50 mg/mL,about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, or about 32mg/mL; about 32 mg/mL to about 50 mg/mL, about 48 mg/mL, about 46 mg/mL,about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36mg/mL, or about 34 mg/mL; about 34 mg/mL to about 50 mg/mL, about 48mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL,about 38 mg/mL, or about 36 mg/mL; about 36 mg/mL to about 50 mg/mL,about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40mg/mL, or about 38 mg/mL; about 38 mg/mL to about 50 mg/mL, about 48mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, or about 40mg/mL; about 40 mg/mL to about 50 mg/mL, about 48 mg/mL, about 46 mg/mL,about 44 mg/mL, or about 42 mg/mL; about 42 mg/mL to about 50 mg/mL,about 48 mg/mL, about 46 mg/mL, or about 44 mg/mL; about 44 mg/mL toabout 50 mg/mL, about 48 mg/mL, or about 46 mg/mL; about 46 mg/mL toabout 50 mg/mL or about 48 mg/mL; or about 48 mg/mL to about 50 mg/mL,recombinant therapeutic protein into the circuit system

In some embodiments, the first period of time can be about 0.1 minute toabout 12 hours, about 11.5 hours, about 11 hours, about 10.5 hours,about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5 hours, about8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours, about 6.0hours, about 5.5 hours, about 5.0 hours, about 4.5 hours, about 4.0hours, about 3.5 hours, about 3.0 hours, about 2.5 hours, about 2.0hours, about 1.5 hours, about 1.0 hour, about 55 minutes, about 50minutes, about 45 minutes, about 40 minutes, about 35 minutes, about 30minutes, about 25 minutes, about 20 minutes, about 15 minutes, about 10minutes, about 5 minutes, about 2 minutes, or about 1 minute; about 1minute to about 12 hours, about 11.5 hours, about 11 hours, about 10.5hours, about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5hours, about 8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours,about 6.0 hours, about 5.5 hours, about 5.0 hours, about 4.5 hours,about 4.0 hours, about 3.5 hours, about 3.0 hours, about 2.5 hours,about 2.0 hours, about 1.5 hours, about 1.0 hour, about 55 minutes,about 50 minutes, about 45 minutes, about 40 minutes, about 35 minutes,about 30 minutes, about 25 minutes, about 20 minutes, about 15 minutes,about 10 minutes, about 5 minutes, or about 2 minutes; about 2 minutesto about 12 hours, about 11.5 hours, about 11 hours, about 10.5 hours,about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5 hours, about8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours, about 6.0hours, about 5.5 hours, about 5.0 hours, about 4.5 hours, about 4.0hours, about 3.5 hours, about 3.0 hours, about 2.5 hours, about 2.0hours, about 1.5 hours, about 1.0 hour, about 55 minutes, about 50minutes, about 45 minutes, about 40 minutes, about 35 minutes, about 30minutes, about 25 minutes, about 20 minutes, about 15 minutes, about 10minutes, or about 5 minutes; about 5 minutes to about 12 hours, about11.5 hours, about 11 hours, about 10.5 hours, about 10 hours, about 9.5hours, about 9.0 hours, about 8.5 hours, about 8.0 hours, about 7.5hours, about 7 hours, about 6.5 hours, about 6.0 hours, about 5.5 hours,about 5.0 hours, about 4.5 hours, about 4.0 hours, about 3.5 hours,about 3.0 hours, about 2.5 hours, about 2.0 hours, about 1.5 hours,about 1.0 hour, about 55 minutes, about 50 minutes, about 45 minutes,about 40 minutes, about 35 minutes, about 30 minutes, about 25 minutes,about 20 minutes, about 15 minutes, or about 10 minutes; about 10minutes to about 12 hours, about 11.5 hours, about 11 hours, about 10.5hours, about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5hours, about 8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours,about 6.0 hours, about 5.5 hours, about 5.0 hours, about 4.5 hours,about 4.0 hours, about 3.5 hours, about 3.0 hours, about 2.5 hours,about 2.0 hours, about 1.5 hours, about 1.0 hour, about 55 minutes,about 50 minutes, about 45 minutes, about 40 minutes, about 35 minutes,about 30 minutes, about 25 minutes, about 20 minutes, or about 15minutes; about 15 minutes to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours,about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.0 hour, about55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, about35 minutes, about 30 minutes, about 25 minutes, or about 20 minutes;about 20 minutes to about 12 hours, about 11.5 hours, about 11 hours,about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0 hours,about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours, about6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours, about 4.5hours, about 4.0 hours, about 3.5 hours, about 3.0 hours, about 2.5hours, about 2.0 hours, about 1.5 hours, about 1.0 hour, about 55minutes, about 50 minutes, about 45 minutes, about 40 minutes, about 35minutes, about 30 minutes, or about 25 minutes; about 25 minutes toabout 12 hours, about 11.5 hours, about 11 hours, about 10.5 hours,about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5 hours, about8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours, about 6.0hours, about 5.5 hours, about 5.0 hours, about 4.5 hours, about 4.0hours, about 3.5 hours, about 3.0 hours, about 2.5 hours, about 2.0hours, about 1.5 hours, about 1.0 hour, about 55 minutes, about 50minutes, about 45 minutes, about 40 minutes, about 35 minutes, or about30 minutes; about 30 minutes to about 12 hours, about 11.5 hours, about11 hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours,about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.0 hour, about55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, orabout 35 minutes; about 35 minutes to about 12 hours, about 11.5 hours,about 11 hours, about 10.5 hours, about 10 hours, about 9.5 hours, about9.0 hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7hours, about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0hours, about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0hours, about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.0hour, about 55 minutes, about 50 minutes, about 45 minutes, or about 40minutes; about 40 minutes to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours,about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.0 hour, about55 minutes, about 50 minutes, or about 45 minutes; about 45 minutes toabout 12 hours, about 11.5 hours, about 11 hours, about 10.5 hours,about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5 hours, about8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours, about 6.0hours, about 5.5 hours, about 5.0 hours, about 4.5 hours, about 4.0hours, about 3.5 hours, about 3.0 hours, about 2.5 hours, about 2.0hours, about 1.5 hours, about 1.0 hour, about 55 minutes, or about 50minutes; about 50 minutes to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours,about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.0 hour, orabout 55 minutes; about 55 minutes to about 12 hours, about 11.5 hours,about 11 hours, about 10.5 hours, about 10 hours, about 9.5 hours, about9.0 hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7hours, about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0hours, about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0hours, about 2.5 hours, about 2.0 hours, about 1.5 hours, or about 1.0hour; about 1.0 hour to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours,about 2.5 hours, about 2.0 hours, or about 1.5 hours; about 1.5 hours toabout 12 hours, about 11.5 hours, about 11 hours, about 10.5 hours,about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5 hours, about8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours, about 6.0hours, about 5.5 hours, about 5.0 hours, about 4.5 hours, about 4.0hours, about 3.5 hours, about 3.0 hours, about 2.5 hours, or about 2.0hours; about 2.0 hours to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours, orabout 2.5 hours; about 2.5 hours to about 12 hours, about 11.5 hours,about 11 hours, about 10.5 hours, about 10 hours, about 9.5 hours, about9.0 hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7hours, about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0hours, about 4.5 hours, about 4.0 hours, about 3.5 hours, or about 3.0hours; about 3.0 hours to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, or about 3.5 hours; about 3.5 hours toabout 12 hours, about 11.5 hours, about 11 hours, about 10.5 hours,about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5 hours, about8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours, about 6.0hours, about 5.5 hours, about 5.0 hours, about 4.5 hours, or about 4.0hours; about 4.0 hours to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours, orabout 4.5 hours; about 4.5 hours to about 12 hours, about 11.5 hours,about 11 hours, about 10.5 hours, about 10 hours, about 9.5 hours, about9.0 hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7hours, about 6.5 hours, about 6.0 hours, about 5.5 hours, or about 5.0hours; about 5.0 hours to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, or about 5.5 hours; about 5.5 hours toabout 12 hours, about 11.5 hours, about 11 hours, about 10.5 hours,about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5 hours, about8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours, or about 6.0hours; about 6.0 hours to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,or about 6.5 hours; about 6.5 hours to about 12 hours, about 11.5 hours,about 11 hours, about 10.5 hours, about 10 hours, about 9.5 hours, about9.0 hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, or about 7hours; about 7 hours to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, or about 7.5 hours; about 7.5hours to about 12 hours, about 11.5 hours, about 11 hours, about 10.5hours, about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5hours, or about 8.0 hours; about 8.0 hours to about 12 hours, about 11.5hours, about 11 hours, about 10.5 hours, about 10 hours, about 9.5hours, about 9.0 hours, or about 8.5 hours; about 8.5 hours to about 12hours, about 11.5 hours, about 11 hours, about 10.5 hours, about 10hours, about 9.5 hours, or about 9.0 hours; about 9.0 hours to about 12hours, about 11.5 hours, about 11 hours, about 10.5 hours, about 10hours, or about 9.5 hours; about 9.5 hours to about 12 hours, about 11.5hours, about 11 hours, about 10.5 hours, or about 10 hours; about 10hours to about 12 hours, about 11.5 hours, about 11 hours, or about 10.5hours; about 10.5 hours to about 12 hours, about 11.5 hours, or about 11hours; about 11 hours to about 12 hours, or about 11.5 hours.

In some examples, step (c) includes continuously flowing (e.g.,unidirectionally or bidirectionally flowing) a diafiltration medium intothe circuit system at a rate of between about 0.05 mL/minute to about100 L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute,about 60 L/minute, about 50 L/minute, about 40 L/minute, about 30L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, about 6.5mL/minute, about 6.0 mL/minute, about 5.5 mL/minute, about 5.0mL/minute, about 4.5 mL/minute, about 4.0 mL/minute, about 3.5mL/minute, about 3.0 mL/minute, about 2.5 mL/minute, about 2.0mL/minute, about 1.5 mL/minute, about 1.0 mL/minute, about 0.5mL/minute, or about 0.1 mL/minute; about 0.1 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,about 800 mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute, about 24 mL/minute, about 22 mL/minute, about 20mL/minute, about 18 mL/minute, about 16 mL/minute, about 14 mL/minute,about 12 mL/minute, about 10 mL/minute, about 9.5 mL/minute, about 9.0mL/minute, about 8.5 mL/minute, about 8.0 mL/minute, about 7.5mL/minute, about 7.0 mL/minute, about 6.5 mL/minute, about 6.0mL/minute, about 5.5 mL/minute, about 5.0 mL/minute, about 4.5mL/minute, about 4.0 mL/minute, about 3.5 mL/minute, about 3.0mL/minute, about 2.5 mL/minute, about 2.0 mL/minute, about 1.5mL/minute, about 1.0 mL/minute, or about 0.5 mL/minute; about 0.5mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, about 6.5mL/minute, about 6.0 mL/minute, about 5.5 mL/minute, about 5.0mL/minute, about 4.5 mL/minute, about 4.0 mL/minute, about 3.5mL/minute, about 3.0 mL/minute, about 2.5 mL/minute, about 2.0mL/minute, about 1.5 mL/minute, or about 1.0 mL/minute; about 1.0mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, about 6.5mL/minute, about 6.0 mL/minute, about 5.5 mL/minute, about 5.0mL/minute, about 4.5 mL/minute, about 4.0 mL/minute, about 3.5mL/minute, about 3.0 mL/minute, about 2.5 mL/minute, about 2.0mL/minute, or about 1.5 mL/minute; about 1.5 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,about 800 mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute, about 24 mL/minute, about 22 mL/minute, about 20mL/minute, about 18 mL/minute, about 16 mL/minute, about 14 mL/minute,about 12 mL/minute, about 10 mL/minute, about 9.5 mL/minute, about 9.0mL/minute, about 8.5 mL/minute, about 8.0 mL/minute, about 7.5mL/minute, about 7.0 mL/minute, about 6.5 mL/minute, about 6.0mL/minute, about 5.5 mL/minute, about 5.0 mL/minute, about 4.5mL/minute, about 4.0 mL/minute, about 3.5 mL/minute, about 3.0mL/minute, about 2.5 mL/minute, or about 2.0 mL/minute; about 2.0mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, about 6.5mL/minute, about 6.0 mL/minute, about 5.5 mL/minute, about 5.0mL/minute, about 4.5 mL/minute, about 4.0 mL/minute, about 3.5mL/minute, about 3.0 mL/minute, or about 2.5 mL/minute; about 2.5mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, about 6.5mL/minute, about 6.0 mL/minute, about 5.5 mL/minute, about 5.0mL/minute, about 4.5 mL/minute, about 4.0 mL/minute, about 3.5mL/minute, or about 3.0 mL/minute; about 3.0 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,about 800 mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute, about 24 mL/minute, about 22 mL/minute, about 20mL/minute, about 18 mL/minute, about 16 mL/minute, about 14 mL/minute,about 12 mL/minute, about 10 mL/minute, about 9.5 mL/minute, about 9.0mL/minute, about 8.5 mL/minute, about 8.0 mL/minute, about 7.5mL/minute, about 7.0 mL/minute, about 6.5 mL/minute, about 6.0mL/minute, about 5.5 mL/minute, about 5.0 mL/minute, about 4.5mL/minute, about 4.0 mL/minute, or about 3.5 mL/minute; about 3.5mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, about 6.5mL/minute, about 6.0 mL/minute, about 5.5 mL/minute, about 5.0mL/minute, about 4.5 mL/minute, or about 4.0 mL/minute; about 4.0mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, about 6.5mL/minute, about 6.0 mL/minute, about 5.5 mL/minute, about 5.0mL/minute, or about 4.5 mL/minute; about 4.5 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,about 800 mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute, about 24 mL/minute, about 22 mL/minute, about 20mL/minute, about 18 mL/minute, about 16 mL/minute, about 14 mL/minute,about 12 mL/minute, about 10 mL/minute, about 9.5 mL/minute, about 9.0mL/minute, about 8.5 mL/minute, about 8.0 mL/minute, about 7.5mL/minute, about 7.0 mL/minute, about 6.5 mL/minute, about 6.0mL/minute, about 5.5 mL/minute, or about 5.0 mL/minute; about 5.0mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, about 6.5mL/minute, about 6.0 mL/minute, or about 5.5 mL/minute; about 5.5mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, about 6.5mL/minute, or about 6.0 mL/minute; about 6.0 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,about 800 mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute, about 24 mL/minute, about 22 mL/minute, about 20mL/minute, about 18 mL/minute, about 16 mL/minute, about 14 mL/minute,about 12 mL/minute, about 10 mL/minute, about 9.5 mL/minute, about 9.0mL/minute, about 8.5 mL/minute, about 8.0 mL/minute, about 7.5mL/minute, about 7.0 mL/minute, or about 6.5 mL/minute; about 6.5mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, or about 7.0 mL/minute; about 7.0mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, or about 7.5 mL/minute; about 7.5 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,about 800 mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute, about 24 mL/minute, about 22 mL/minute, about 20mL/minute, about 18 mL/minute, about 16 mL/minute, about 14 mL/minute,about 12 mL/minute, about 10 mL/minute, about 9.5 mL/minute, about 9.0mL/minute, about 8.5 mL/minute, or about 8.0 mL/minute; about 8.0mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, or about 8.5 mL/minute; about8.5 mL/minute to about 100 L/minute, about 90 L/minute, about 80L/minute, about 70 L/minute, about 60 L/minute, about 50 L/minute, about40 L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute,about 5 L/minute, about 1 L/minute, about 950 mL/minute, about 900mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, or about 9.0 mL/minute; about9.0 mL/minute to about 100 L/minute, about 90 L/minute, about 80L/minute, about 70 L/minute, about 60 L/minute, about 50 L/minute, about40 L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute,about 5 L/minute, about 1 L/minute, about 950 mL/minute, about 900mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, or about 9.5 mL/minute; about 9.5 mL/minute to about100 L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute,about 60 L/minute, about 50 L/minute, about 40 L/minute, about 30L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, or about 10mL/minute; about 10 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, or about 12mL/minute; about 12 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, or about 14 mL/minute; about 14 mL/minuteto about 100 L/minute, about 90 L/minute, about 80 L/minute, about 70L/minute, about 60 L/minute, about 50 L/minute, about 40 L/minute, about30 L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute,about 1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, or about 16mL/minute; about 16 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, or about 18mL/minute; about 18 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, or about 20 mL/minute; about 20mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,or about 22 mL/minute; about 22 mL/minute to about 100 L/minute, about90 L/minute, about 80 L/minute, about 70 L/minute, about 60 L/minute,about 50 L/minute, about 40 L/minute, about 30 L/minute, about 20L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about950 mL/minute, about 900 mL/minute, about 850 mL/minute, about 800mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute, or about 24 mL/minute; about 23 mL/minute to about100 L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute,about 60 L/minute, about 50 L/minute, about 40 L/minute, about 30L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, or about 25 mL/minute; about 25 mL/minuteto about 100 L/minute, about 90 L/minute, about 80 L/minute, about 70L/minute, about 60 L/minute, about 50 L/minute, about 40 L/minute, about30 L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute,about 1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, or about 50 mL/minute; about 50 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,about 800 mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, or about 100 mL/minute; about 100mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, or about 150 mL/minute; about 150mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, or about 200 mL/minute; about 200 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,about 800 mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, or about 250 mL/minute; about 250mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, or about 300 mL/minute; about 300mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, or about 350 mL/minute; about 350 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,about 800 mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, or about 400 mL/minute; about 400mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, or about 450 mL/minute; about 450mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, or about 500 mL/minute; about 500 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,about 800 mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, or about 550 mL/minute; about 550mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, or about 600 mL/minute; about 600mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, or about 650 mL/minute; about 650 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,about 800 mL/minute, about 750 mL/minute, or about 700 mL/minute; about700 mL/minute to about 100 L/minute, about 90 L/minute, about 80L/minute, about 70 L/minute, about 60 L/minute, about 50 L/minute, about40 L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute,about 5 L/minute, about 1 L/minute, about 950 mL/minute, about 900mL/minute, about 850 mL/minute, about 800 mL/minute, or about 750mL/minute; about 750 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, or about 800 mL/minute; about800 mL/minute to about 100 L/minute, about 90 L/minute, about 80L/minute, about 70 L/minute, about 60 L/minute, about 50 L/minute, about40 L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute,about 5 L/minute, about 1 L/minute, about 950 mL/minute, about 900mL/minute, or about 850 mL/minute; about 850 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, or about 900 mL/minute; about 900mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, or about 950 mL/minute; about 950mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, or about 1 L/minute; about 1 L/minute to about 100 L/minute,about 90 L/minute, about 80 L/minute, about 70 L/minute, about 60L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute, about20 L/minute, about 10 L/minute, or about 5 L/minute; about 5 L/minute toabout 100 L/minute, about 90 L/minute, about 80 L/minute, about 70L/minute, about 60 L/minute, about 50 L/minute, about 40 L/minute, about30 L/minute, about 20 L/minute, or about 10 L/minute; about 10 L/minuteto about 100 L/minute, about 90 L/minute, about 80 L/minute, about 70L/minute, about 60 L/minute, about 50 L/minute, about 40 L/minute, about30 L/minute, or about 20 L/minute; about 20 L/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, or about 30 L/minute;about 30 L/minute to about 100 L/minute, about 90 L/minute, about 80L/minute, about 70 L/minute, about 60 L/minute, about 50 L/minute, orabout 40 L/minute; about 40 L/minute to about 100 L/minute, about 90L/minute, about 80 L/minute, about 70 L/minute, about 60 L/minute, orabout 50 L/minute; about 50 L/minute to about 100 L/minute, about 90L/minute, about 80 L/minute, about 70 L/minute, or about 60 L/minute;about 60 L/minute to about 100 L/minute, about 90 L/minute, about 80L/minute, or about 70 L/minute; about 70 L/minute to about 100 L/minute,about 90 L/minute, or about 80 L/minute; about 80 L/minute to about 100L/minute or about 90 L/minute; or about 90 L/minute to about 100L/minute.

A diafiltration medium can be, e.g., a physiologically acceptable buffer(e.g., phosphate buffered saline or a buffer used to formulate therecombinant therapeutic protein, e.g., a buffer comprising one or moreof glycine, phosphate, acetate, citrate, a bulking agent (e.g., anon-reducing carbohydrate, e.g., sucrose), a surfactant (e.g.,polysorbate 80, Triton X-100, and Tween).

In some examples of these methods, the second period of time can beabout 0.1 minute to about 12 hours, about 11.5 hours, about 11 hours,about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0 hours,about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours, about6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours, about 4.5hours, about 4.0 hours, about 3.5 hours, about 3.0 hours, about 2.5hours, about 2.0 hours, about 1.5 hours, about 1.0 hour, about 55minutes, about 50 minutes, about 45 minutes, about 40 minutes, about 35minutes, about 30 minutes, about 25 minutes, about 20 minutes, about 15minutes, about 10 minutes, about 5 minutes, about 2 minutes, or about 1minute; about 1 minute to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours,about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.0 hour, about55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, about35 minutes, about 30 minutes, about 25 minutes, about 20 minutes, about15 minutes, about 10 minutes, about 5 minutes, or about 2 minutes; about2 minutes to about 12 hours, about 11.5 hours, about 11 hours, about10.5 hours, about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5hours, about 8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours,about 6.0 hours, about 5.5 hours, about 5.0 hours, about 4.5 hours,about 4.0 hours, about 3.5 hours, about 3.0 hours, about 2.5 hours,about 2.0 hours, about 1.5 hours, about 1.0 hour, about 55 minutes,about 50 minutes, about 45 minutes, about 40 minutes, about 35 minutes,about 30 minutes, about 25 minutes, about 20 minutes, about 15 minutes,about 10 minutes, or about 5 minutes; about 5 minutes to about 12 hours,about 11.5 hours, about 11 hours, about 10.5 hours, about 10 hours,about 9.5 hours, about 9.0 hours, about 8.5 hours, about 8.0 hours,about 7.5 hours, about 7 hours, about 6.5 hours, about 6.0 hours, about5.5 hours, about 5.0 hours, about 4.5 hours, about 4.0 hours, about 3.5hours, about 3.0 hours, about 2.5 hours, about 2.0 hours, about 1.5hours, about 1.0 hour, about 55 minutes, about 50 minutes, about 45minutes, about 40 minutes, about 35 minutes, about 30 minutes, about 25minutes, about 20 minutes, about 15 minutes, or about 10 minutes; about10 minutes to about 12 hours, about 11.5 hours, about 11 hours, about10.5 hours, about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5hours, about 8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours,about 6.0 hours, about 5.5 hours, about 5.0 hours, about 4.5 hours,about 4.0 hours, about 3.5 hours, about 3.0 hours, about 2.5 hours,about 2.0 hours, about 1.5 hours, about 1.0 hour, about 55 minutes,about 50 minutes, about 45 minutes, about 40 minutes, about 35 minutes,about 30 minutes, about 25 minutes, about 20 minutes, or about 15minutes; about 15 minutes to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours,about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.0 hour, about55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, about35 minutes, about 30 minutes, about 25 minutes, or about 20 minutes;about 20 minutes to about 12 hours, about 11.5 hours, about 11 hours,about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0 hours,about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours, about6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours, about 4.5hours, about 4.0 hours, about 3.5 hours, about 3.0 hours, about 2.5hours, about 2.0 hours, about 1.5 hours, about 1.0 hour, about 55minutes, about 50 minutes, about 45 minutes, about 40 minutes, about 35minutes, about 30 minutes, or about 25 minutes; about 25 minutes toabout 12 hours, about 11.5 hours, about 11 hours, about 10.5 hours,about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5 hours, about8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours, about 6.0hours, about 5.5 hours, about 5.0 hours, about 4.5 hours, about 4.0hours, about 3.5 hours, about 3.0 hours, about 2.5 hours, about 2.0hours, about 1.5 hours, about 1.0 hour, about 55 minutes, about 50minutes, about 45 minutes, about 40 minutes, about 35 minutes, or about30 minutes; about 30 minutes to about 12 hours, about 11.5 hours, about11 hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours,about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.0 hour, about55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, orabout 35 minutes; about 35 minutes to about 12 hours, about 11.5 hours,about 11 hours, about 10.5 hours, about 10 hours, about 9.5 hours, about9.0 hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7hours, about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0hours, about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0hours, about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.0hour, about 55 minutes, about 50 minutes, about 45 minutes, or about 40minutes; about 40 minutes to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours,about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.0 hour, about55 minutes, about 50 minutes, or about 45 minutes; about 45 minutes toabout 12 hours, about 11.5 hours, about 11 hours, about 10.5 hours,about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5 hours, about8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours, about 6.0hours, about 5.5 hours, about 5.0 hours, about 4.5 hours, about 4.0hours, about 3.5 hours, about 3.0 hours, about 2.5 hours, about 2.0hours, about 1.5 hours, about 1.0 hour, about 55 minutes, or about 50minutes; about 50 minutes to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours,about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.0 hour, orabout 55 minutes; about 55 minutes to about 12 hours, about 11.5 hours,about 11 hours, about 10.5 hours, about 10 hours, about 9.5 hours, about9.0 hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7hours, about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0hours, about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0hours, about 2.5 hours, about 2.0 hours, about 1.5 hours, or about 1.0hour; about 1.0 hour to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours,about 2.5 hours, about 2.0 hours, or about 1.5 hours; about 1.5 hours toabout 12 hours, about 11.5 hours, about 11 hours, about 10.5 hours,about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5 hours, about8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours, about 6.0hours, about 5.5 hours, about 5.0 hours, about 4.5 hours, about 4.0hours, about 3.5 hours, about 3.0 hours, about 2.5 hours, or about 2.0hours; about 2.0 hours to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours, orabout 2.5 hours; about 2.5 hours to about 12 hours, about 11.5 hours,about 11 hours, about 10.5 hours, about 10 hours, about 9.5 hours, about9.0 hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7hours, about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0hours, about 4.5 hours, about 4.0 hours, about 3.5 hours, or about 3.0hours; about 3.0 hours to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, or about 3.5 hours; about 3.5 hours toabout 12 hours, about 11.5 hours, about 11 hours, about 10.5 hours,about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5 hours, about8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours, about 6.0hours, about 5.5 hours, about 5.0 hours, about 4.5 hours, or about 4.0hours; about 4.0 hours to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours, orabout 4.5 hours; about 4.5 hours to about 12 hours, about 11.5 hours,about 11 hours, about 10.5 hours, about 10 hours, about 9.5 hours, about9.0 hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7hours, about 6.5 hours, about 6.0 hours, about 5.5 hours, or about 5.0hours; about 5.0 hours to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, or about 5.5 hours; about 5.5 hours toabout 12 hours, about 11.5 hours, about 11 hours, about 10.5 hours,about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5 hours, about8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours, or about 6.0hours; about 6.0 hours to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,or about 6.5 hours; about 6.5 hours to about 12 hours, about 11.5 hours,about 11 hours, about 10.5 hours, about 10 hours, about 9.5 hours, about9.0 hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, or about 7hours; about 7 hours to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, or about 7.5 hours; about 7.5hours to about 12 hours, about 11.5 hours, about 11 hours, about 10.5hours, about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5hours, or about 8.0 hours; about 8.0 hours to about 12 hours, about 11.5hours, about 11 hours, about 10.5 hours, about 10 hours, about 9.5hours, about 9.0 hours, or about 8.5 hours; about 8.5 hours to about 12hours, about 11.5 hours, about 11 hours, about 10.5 hours, about 10hours, about 9.5 hours, or about 9.0 hours; about 9.0 hours to about 12hours, about 11.5 hours, about 11 hours, about 10.5 hours, about 10hours, or about 9.5 hours; about 9.5 hours to about 12 hours, about 11.5hours, about 11 hours, about 10.5 hours, or about 10 hours; about 10hours to about 12 hours, about 11.5 hours, about 11 hours, or about 10.5hours; about 10.5 hours to about 12 hours, about 11.5 hours, or about 11hours; about 11 hours to about 12 hours, or about 11.5 hours.

In some embodiments, the fluid including the recombinant therapeuticprotein that is collected in step (d) includes a concentration ofrecombinant therapeutic protein of about 0.1 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL,about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL,about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL,about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL, about 18mg/mL, about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, about 10 mg/mL,about 9 mg/mL, about 8 mg/mL, about 7 mg/mL, about 6 mg/mL, about 5mg/mL, about 4 mg/mL, about 3 mg/mL, about 2.5 mg/mL, about 2.0 mg/mL,about 1.5 mg/mL, about 1.0 mg/mL, or about 0.5 mg/mL; about 0.5 mg/mL toabout 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL,about 64 mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56mg/mL, about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL,about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL,about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20mg/mL, about 18 mg/mL, about 16 mg/mL, about 14 mg/mL, about 12 mg/mL,about 10 mg/mL, about 9 mg/mL, about 8 mg/mL, about 7 mg/mL, about 6mg/mL, about 5 mg/mL, about 4 mg/mL, about 3 mg/mL, about 2.5 mg/mL,about 2.0 mg/mL, about 1.5 mg/mL, or about 1.0 mg/mL; about 1.0 mg/mL toabout 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL,about 64 mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56mg/mL, about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL,about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL,about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20mg/mL, about 18 mg/mL, about 16 mg/mL, about 14 mg/mL, about 12 mg/mL,about 10 mg/mL, about 9 mg/mL, about 8 mg/mL, about 7 mg/mL, about 6mg/mL, about 5 mg/mL, about 4 mg/mL, about 3 mg/mL, about 2.5 mg/mL,about 2.0 mg/mL, or about 1.5 mg/mL; about 1.5 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL,about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL,about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL,about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL, about 18mg/mL, about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, about 10 mg/mL,about 9 mg/mL, about 8 mg/mL, about 7 mg/mL, about 6 mg/mL, about 5mg/mL, about 4 mg/mL, about 3 mg/mL, about 2.5 mg/mL, or about 2.0mg/mL; about 2.0 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL,about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL,about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL,about 22 mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14mg/mL, about 12 mg/mL, about 10 mg/mL, about 9 mg/mL, about 8 mg/mL,about 7 mg/mL, about 6 mg/mL, about 5 mg/mL, about 4 mg/mL, about 3mg/mL, or about 2.5 mg/mL; about 2.5 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72 mg/mL,about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL, about 62mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54 mg/mL,about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL,about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL, about 26mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL, about 18 mg/mL,about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, about 10 mg/mL, about 9mg/mL, about 8 mg/mL, about 7 mg/mL, about 6 mg/mL, about 5 mg/mL, about4 mg/mL, or about 3 mg/mL; about 3 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72 mg/mL,about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL, about 62mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54 mg/mL,about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL,about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL, about 26mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL, about 18 mg/mL,about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, about 10 mg/mL, about 9mg/mL, about 8 mg/mL, about 7 mg/mL, about 6 mg/mL, about 5 mg/mL, orabout 4 mg/mL; about 4 mg/mL to about 100 mg/mL, about 98 mg/mL, 96mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL,about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78mg/mL, about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL,about 68 mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60mg/mL, about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL,about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL,about 32 mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24mg/mL, about 22 mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL,about 14 mg/mL, about 12 mg/mL, about 10 mg/mL, about 9 mg/mL, about 8mg/mL, about 7 mg/mL, about 6 mg/mL, or about 5 mg/mL; about 5 mg/mL toabout 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL,about 64 mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56mg/mL, about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL,about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL,about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20mg/mL, about 18 mg/mL, about 16 mg/mL, about 14 mg/mL, about 12 mg/mL,about 10 mg/mL, about 9 mg/mL, about 8 mg/mL, about 7 mg/mL, or about 6mg/mL; about 6 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL, about94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL,about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL,about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL,about 22 mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14mg/mL, about 12 mg/mL, about 10 mg/mL, about 9 mg/mL, about 8 mg/mL, orabout 7 mg/mL; about 7 mg/mL to about 100 mg/mL, about 98 mg/mL, 96mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL,about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78mg/mL, about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL,about 68 mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60mg/mL, about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL,about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL,about 32 mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24mg/mL, about 22 mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL,about 14 mg/mL, about 12 mg/mL, about 10 mg/mL, about 9 mg/mL, or about8 mg/mL; about 8 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL,about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL,about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL,about 22 mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14mg/mL, about 12 mg/mL, about 10 mg/mL, or about 9 mg/mL; about 9 mg/mLto about 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL,about 64 mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56mg/mL, about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL,about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL,about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20mg/mL, about 18 mg/mL, about 16 mg/mL, about 14 mg/mL, about 12 mg/mL,or about 10 mg/mL; about 10 mg/mL to about 100 mg/mL, about 98 mg/mL, 96mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL,about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78mg/mL, about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL,about 68 mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60mg/mL, about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL,about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL,about 32 mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24mg/mL, about 22 mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL,about 14 mg/mL, or about 12 mg/mL; about 12 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL,about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL,about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL,about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL, about 18mg/mL, about 16 mg/mL, or about 14 mg/mL; about 14 mg/mL to about 100mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL,about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL,about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL,about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL,about 18 mg/mL, or about 16 mg/mL; about 16 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL,about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL,about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL,about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL, or about18 mg/mL; about 18 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL,about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL,about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL,about 22 mg/mL, or about 20 mg/mL; about 20 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL,about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL,about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL,about 26 mg/mL, about 24 mg/mL, or about 22 mg/mL; about 22 mg/mL toabout 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL,about 64 mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56mg/mL, about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL,about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL,about 28 mg/mL, about 26 mg/mL, or about 24 mg/mL; about 24 mg/mL toabout 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL,about 64 mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56mg/mL, about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL,about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL,about 28 mg/mL, or about 26 mg/mL; about 26 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL,about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL,about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, or about 28mg/mL; about 28 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL,about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL,about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32mg/mL, or about 30 mg/mL; about 30 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72 mg/mL,about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL, about 62mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54 mg/mL,about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL,about 34 mg/mL, or about 32 mg/mL; about 32 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL,about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL,about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36mg/mL, or about 34 mg/mL; about 34 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72 mg/mL,about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL, about 62mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54 mg/mL,about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, or about 36mg/mL; about 36 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL,about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL,about 40 mg/mL, or about 38 mg/mL; about 38 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL,about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL,about 44 mg/mL, about 42 mg/mL, or about 40 mg/mL; about 40 mg/mL toabout 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL,about 64 mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56mg/mL, about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL,about 46 mg/mL, about 44 mg/mL, or about 42 mg/mL; about 42 mg/mL toabout 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL,about 64 mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56mg/mL, about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL,about 46 mg/mL, or about 44 mg/mL; about 44 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL,about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, or about 46mg/mL; about 46 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL,about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50mg/mL, or about 48 mg/mL; about 48 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72 mg/mL,about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL, about 62mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54 mg/mL,about 52 mg/mL, or about 50 mg/mL; about 50 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL,about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54mg/mL, or about 52 mg/mL; about 52 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72 mg/mL,about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL, about 62mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, or about 54mg/mL; about 54 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL,about 58 mg/mL, or about 56 mg/mL; about 56 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL,about 62 mg/mL, about 60 mg/mL, or about 58 mg/mL; about 58 mg/mL toabout 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL,about 64 mg/mL, about 62 mg/mL, or about 60 mg/mL; about 60 mg/mL toabout 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL,about 64 mg/mL, or about 62 mg/mL; about 62 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, or about 64mg/mL; about 64 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, or about 66 mg/mL; about 66 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72 mg/mL,about 70 mg/mL, or about 68 mg/mL; about 68 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, or about 70 mg/mL; about 70 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, or about 72mg/mL; about 72 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, or about 74 mg/mL; about 74 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, or about 76 mg/mL; about 76 mg/mL toabout 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, about 80 mg/mL, or about 78 mg/mL; about 78 mg/mL toabout 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, or about 80 mg/mL; about 80 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, or about 82mg/mL; about 82 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, or about 84 mg/mL; about 84 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about88 mg/mL, or about 86 mg/mL; about 86 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, orabout 88 mg/mL; about 88 mg/mL to about 100 mg/mL, about 98 mg/mL, 96mg/mL, about 94 mg/mL, about 92 mg/mL, or about 90 mg/mL; about 90 mg/mLto about 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, or about92 mg/mL; about 92 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,or about 94 mg/mL; about 94 mg/mL to about 100 mg/mL, about 98 mg/mL, or96 mg/mL; about 96 mg/mL to about 100 mg/mL or about 98 mg/mL; or about98 mg/mL to about 100 mg/mL.

In some embodiments, these fold-diafiltration achieved after the firstand second time periods is about 1-fold to about 20-fold, about 19-fold,about 18-fold, about 17-fold, about 16-fold, about 15-fold, about14-fold, about 13-fold, about 12-fold, about 11-fold, about 10-fold,about 9-fold, about 8-fold, about 7-fold, about 6-fold, about 5-fold,about 4-fold, about 3-fold, or about 2-fold; about 2-fold to about20-fold, about 19-fold, about 18-fold, about 17-fold, about 16-fold,about 15-fold, about 14-fold, about 13-fold, about 12-fold, about11-fold, about 10-fold, about 9-fold, about 8-fold, about 7-fold, about6-fold, about 5-fold, about 4-fold, or about 3-fold; about 3-fold toabout 20-fold, about 19-fold, about 18-fold, about 17-fold, about16-fold, about 15-fold, about 14-fold, about 13-fold, about 12-fold,about 11-fold, about 10-fold, about 9-fold, about 8-fold, about 7-fold,about 6-fold, about 5-fold, or about 4-fold; about 4-fold to about20-fold, about 19-fold, about 18-fold, about 17-fold, about 16-fold,about 15-fold, about 14-fold, about 13-fold, about 12-fold, about11-fold, about 10-fold, about 9-fold, about 8-fold, about 7-fold, about6-fold, or about 5-fold; about 5-fold to about 20-fold, about 19-fold,about 18-fold, about 17-fold, about 16-fold, about 15-fold, about14-fold, about 13-fold, about 12-fold, about 11-fold, about 10-fold,about 9-fold, about 8-fold, about 7-fold, or about 6-fold; about 6-foldto about 20-fold, about 19-fold, about 18-fold, about 17-fold, about16-fold, about 15-fold, about 14-fold, about 13-fold, about 12-fold,about 11-fold, about 10-fold, about 9-fold, about 8-fold, or about7-fold; about 7-fold to about 20-fold, about 19-fold, about 18-fold,about 17-fold, about 16-fold, about 15-fold, about 14-fold, about13-fold, about 12-fold, about 11-fold, about 10-fold, about 9-fold, orabout 8-fold; about 8-fold to about 20-fold, about 19-fold, about18-fold, about 17-fold, about 16-fold, about 15-fold, about 14-fold,about 13-fold, about 12-fold, about 11-fold, about 10-fold, or about9-fold; about 9-fold to about 20-fold, about 19-fold, about 18-fold,about 17-fold, about 16-fold, about 15-fold, about 14-fold, about13-fold, about 12-fold, about 11-fold, or about 10-fold; about 10-foldto about 20-fold, about 19-fold, about 18-fold, about 17-fold, about16-fold, about 15-fold, about 14-fold, about 13-fold, about 12-fold, orabout 11-fold; about 11-fold to about 20-fold, about 19-fold, about18-fold, about 17-fold, about 16-fold, about 15-fold, about 14-fold,about 13-fold, or about 12-fold; about 12-fold to about 20-fold, about19-fold, about 18-fold, about 17-fold, about 16-fold, about 15-fold,about 14-fold, or about 13-fold; about 13-fold to about 20-fold, about19-fold, about 18-fold, about 17-fold, about 16-fold, about 15-fold, orabout 14-fold; about 14-fold to about 20-fold, about 19-fold, about18-fold, about 17-fold, about 16-fold, or about 15-fold; about 15-foldto about 20-fold, about 19-fold, about 18-fold, about 17-fold, or about16-fold; about 16-fold to about 20-fold, about 19-fold, about 18-fold,or about 17-fold; about 17-fold to about 20-fold, about 19-fold, orabout 18-fold; about 18-fold to about 20-fold or about 19-fold; or about19-fold to about 20-fold.

In some embodiments, the circuit system does not include a holding tank.

In other embodiments of this method, during the first period of time, afluid including the recombinant therapeutic protein and thediafiltration medium are continuously flowed together into the circuitsystem, and then during the second period of time, only thediafiltration medium is continuously flowed into the circuit system.

The fluid can be collected in step (d) using a syringe, gravity, or apump (e.g., a peristaltic pump disposed in the conduit). Additionalmeans for collecting the fluid including the recombinant therapeuticprotein in step (d) are known in the art.

Some examples of these methods further include: (e) providing a secondcircuit system including (i) a tangential flow virus filtration (TFVF)unit having first and second inlets, and (ii) a conduit in fluidcommunication between the first and second inlets of the TFVF unit,including at least one port for flowing fluid into or out of, or both,of the second system, where the second system is configured such thatfluid can be flowed through the conduit and the TFVF unit, and filtrateincluding the recombinant therapeutic protein can be collected from theTFVF unit; and (f) flowing the collected fluid of step (d) into thesecond system using a connecting conduit in fluid communication betweena port in the system and a port in the second system, and collectingfiltrate including the recombinant therapeutic protein from the TFVFunit for a third period of time. As can be appreciated by those skilledin the art, some embodiments of these methods can further includerecirculating any fluid including the recombinant therapeutic protein inthe conduit of the second circuit system (including a TFVF unit) thatdid not pass through the one or more tangential virus filters in theTFVF unit (e.g., acts as a buffer when a new volume of a fluid includingthe recombinant therapeutic protein is flowed into the conduit of thecircuit system including a TFVF unit).

In some embodiments, the TFVF unit includes one, two, three, four, five,six, seven, eight, nine, ten, eleven, or twelve tangential virusfilters. In some examples of these methods, the TFVF unit can includeone or more tangential virus filter(s) having a surface area of about0.05 cm² to about 100 m², about 90 m², about 80 m², about 70 m², about60 m², about 50 m², about 45 m², about 40 m², about 35 m², about 30 m²,about 25 m², about 20 m², about 15 m², about 10 m², about 5 m², about 1m², about 800 cm², about 600 cm², about 400 cm², about 200 cm², about150 cm², about 100 cm², about 50 cm², about 48 cm², about 46 cm², about44 cm², about 42 cm², about 40 cm², about 38 cm², about 36 cm², about 34cm², about 32 cm², about 30 cm², about 28 cm², about 26 cm², about 24cm², about 22 cm², about 20 cm², about 18 cm², about 16 cm², about 14cm², about 12 cm², about 10 cm², about 9.5 cm², about 9.0 cm², about 8.5cm², about 8.0 cm², about 7.5 cm², about 7.0 cm², about 6.5 cm², about6.0 cm², about 5.5 cm², about 5.0 cm², about 4.5 cm², about 4.0 cm²,about 3.5 cm², about 3.0 cm², about 2.5 cm², about 2.0 cm², about 1.5cm², about 1.0 cm², about 0.5 cm², or about 0.1 cm²; about 0.1 cm² toabout 100 m², about 90 m², about 80 m², about 70 m², about 60 m², about50 m², about 45 m², about 40 m², about 35 m², about 30 m², about 25 m²,about 20 m², about 15 m², about 10 m², about 5 m², about 1 m², about 800cm², about 600 cm², about 400 cm², about 200 cm², about 150 cm², about100 cm², about 50 cm², about 48 cm², about 46 cm², about 44 cm², about42 cm², about 40 cm², about 38 cm², about 36 cm², about 34 cm², about 32cm², about 30 cm², about 28 cm², about 26 cm², about 24 cm², about 22cm², about 20 cm², about 18 cm², about 16 cm², about 14 cm², about 12cm², about 10 cm², about 9.5 cm², about 9.0 cm², about 8.5 cm², about8.0 cm², about 7.5 cm², about 7.0 cm², about 6.5 cm², about 6.0 cm²,about 5.5 cm², about 5.0 cm², about 4.5 cm², about 4.0 cm², about 3.5cm², about 3.0 cm², about 2.5 cm², about 2.0 cm², about 1.5 cm², about1.0 cm², or about 0.5 cm²; about 0.5 cm² to about 100 m², about 90 m²,about 80 m², about 70 m², about 60 m², about 50 m², about 45 m², about40 m², about 35 m², about 30 m², about 25 m², about 20 m², about 15 m²,about 10 m², about 5 m², about 1 m², about 800 cm², about 600 cm², about400 cm², about 200 cm², about 150 cm², about 100 cm², about 50 cm²,about 48 cm², about 46 cm², about 44 cm², about 42 cm², about 40 cm²,about 38 cm², about 36 cm², about 34 cm², about 32 cm², about 30 cm²,about 28 cm², about 26 cm², about 24 cm², about 22 cm², about 20 cm²,about 18 cm², about 16 cm², about 14 cm², about 12 cm², about 10 cm²,about 9.5 cm², about 9.0 cm², about 8.5 cm², about 8.0 cm², about 7.5cm², about 7.0 cm², about 6.5 cm², about 6.0 cm², about 5.5 cm², about5.0 cm², about 4.5 cm², about 4.0 cm², about 3.5 cm², about 3.0 cm²,about 2.5 cm², about 2.0 cm², about 1.5 cm², or about 1.0 cm²; about 1.0cm² to about 100 m², about 90 m², about 80 m², about 70 m², about 60 m²,about 50 m², about 45 m², about 40 m², about 35 m², about 30 m², about25 m², about 20 m², about 15 m², about 10 m², about 5 m², about 1 m²,about 800 cm², about 600 cm², about 400 cm², about 200 cm², about 150cm², about 100 cm², about 50 cm², about 48 cm², about 46 cm², about 44cm², about 42 cm², about 40 cm², about 38 cm², about 36 cm², about 34cm², about 32 cm², about 30 cm², about 28 cm², about 26 cm², about 24cm², about 22 cm², about 20 cm², about 18 cm², about 16 cm², about 14cm², about 12 cm², about 10 cm², about 9.5 cm², about 9.0 cm², about 8.5cm², about 8.0 cm², about 7.5 cm², about 7.0 cm², about 6.5 cm², about6.0 cm², about 5.5 cm², about 5.0 cm², about 4.5 cm², about 4.0 cm²,about 3.5 cm², about 3.0 cm², about 2.5 cm², about 2.0 cm², or about 1.5cm²; about 1.5 cm² to about 100 m², about 90 m², about 80 m², about 70m², about 60 m², about 50 m², about 45 m², about 40 m², about 35 m²,about 30 m², about 25 m², about 20 m², about 15 m², about 10 m², about 5m², about 1 m², about 800 cm², about 600 cm², about 400 cm², about 200cm², about 150 cm², about 100 cm², about 50 cm², about 48 cm², about 46cm², about 44 cm², about 42 cm², about 40 cm², about 38 cm², about 36cm², about 34 cm², about 32 cm², about 30 cm², about 28 cm², about 26cm², about 24 cm², about 22 cm², about 20 cm², about 18 cm², about 16cm², about 14 cm², about 12 cm², about 10 cm², about 9.5 cm², about 9.0cm², about 8.5 cm², about 8.0 cm², about 7.5 cm², about 7.0 cm², about6.5 cm², about 6.0 cm², about 5.5 cm², about 5.0 cm², about 4.5 cm²,about 4.0 cm², about 3.5 cm², about 3.0 cm², about 2.5 cm², or about 2.0cm²; about 2.0 cm² to about 100 m², about 90 m², about 80 m², about 70m², about 60 m², about 50 m², about 45 m², about 40 m², about 35 m²,about 30 m², about 25 m², about 20 m², about 15 m², about 10 m², about 5m², about 1 m², about 800 cm², about 600 cm², about 400 cm², about 200cm², about 150 cm², about 100 cm², about 50 cm², about 48 cm², about 46cm², about 44 cm², about 42 cm², about 40 cm², about 38 cm², about 36cm², about 34 cm², about 32 cm², about 30 cm², about 28 cm², about 26cm², about 24 cm², about 22 cm², about 20 cm², about 18 cm², about 16cm², about 14 cm², about 12 cm², about 10 cm², about 9.5 cm², about 9.0cm², about 8.5 cm², about 8.0 cm², about 7.5 cm², about 7.0 cm², about6.5 cm², about 6.0 cm², about 5.5 cm², about 5.0 cm², about 4.5 cm²,about 4.0 cm², about 3.5 cm², about 3.0 cm², or about 2.5 cm²; about 2.5cm² to about 100 m², about 90 m², about 80 m², about 70 m², about 60 m²,about 50 m², about 45 m², about 40 m², about 35 m², about 30 m², about25 m², about 20 m², about 15 m², about 10 m², about 5 m², about 1 m²,about 800 cm², about 600 cm², about 400 cm², about 200 cm², about 150cm², about 100 cm², about 50 cm², about 48 cm², about 46 cm², about 44cm², about 42 cm², about 40 cm², about 38 cm², about 36 cm², about 34cm², about 32 cm², about 30 cm², about 28 cm², about 26 cm², about 24cm², about 22 cm², about 20 cm², about 18 cm², about 16 cm², about 14cm², about 12 cm², about 10 cm², about 9.5 cm², about 9.0 cm², about 8.5cm², about 8.0 cm², about 7.5 cm², about 7.0 cm², about 6.5 cm², about6.0 cm², about 5.5 cm², about 5.0 cm², about 4.5 cm², about 4.0 cm²,about 3.5 cm², or about 3.0 cm²; about 3.0 cm² to about 100 m², about 90m², about 80 m², about 70 m², about 60 m², about 50 m², about 45 m²,about 40 m², about 35 m², about 30 m², about 25 m², about 20 m², about15 m², about 10 m², about 5 m², about 1 m², about 800 cm², about 600cm², about 400 cm², about 200 cm², about 150 cm², about 100 cm², about50 cm², about 48 cm², about 46 cm², about 44 cm², about 42 cm², about 40cm², about 38 cm², about 36 cm², about 34 cm², about 32 cm², about 30cm², about 28 cm², about 26 cm², about 24 cm², about 22 cm², about 20cm², about 18 cm², about 16 cm², about 14 cm², about 12 cm², about 10cm², about 9.5 cm², about 9.0 cm², about 8.5 cm², about 8.0 cm², about7.5 cm², about 7.0 cm², about 6.5 cm², about 6.0 cm², about 5.5 cm²,about 5.0 cm², about 4.5 cm², about 4.0 cm², or about 3.5 cm²; about 3.5cm² to about 100 m², about 90 m², about 80 m², about 70 m², about 60 m²,about 50 m², about 45 m², about 40 m², about 35 m², about 30 m², about25 m², about 20 m², about 15 m², about 10 m², about 5 m², about 1 m²,about 800 cm², about 600 cm², about 400 cm², about 200 cm², about 150cm², about 100 cm², about 50 cm², about 48 cm², about 46 cm², about 44cm², about 42 cm², about 40 cm², about 38 cm², about 36 cm², about 34cm², about 32 cm², about 30 cm², about 28 cm², about 26 cm², about 24cm², about 22 cm², about 20 cm², about 18 cm², about 16 cm², about 14cm², about 12 cm², about 10 cm², about 9.5 cm², about 9.0 cm², about 8.5cm², about 8.0 cm², about 7.5 cm², about 7.0 cm², about 6.5 cm², about6.0 cm², about 5.5 cm², about 5.0 cm², about 4.5 cm², or about 4.0 cm²;about 4.0 cm² to about 100 m², about 90 m², about 80 m², about 70 m²,about 60 m², about 50 m², about 45 m², about 40 m², about 35 m², about30 m², about 25 m², about 20 m², about 15 m², about 10 m², about 5 m²,about 1 m², about 800 cm², about 600 cm², about 400 cm², about 200 cm²,about 150 cm², about 100 cm², about 50 cm², about 48 cm², about 46 cm²,about 44 cm², about 42 cm², about 40 cm², about 38 cm², about 36 cm²,about 34 cm², about 32 cm², about 30 cm², about 28 cm², about 26 cm²,about 24 cm², about 22 cm², about 20 cm², about 18 cm², about 16 cm²,about 14 cm², about 12 cm², about 10 cm², about 9.5 cm², about 9.0 cm²,about 8.5 cm², about 8.0 cm², about 7.5 cm², about 7.0 cm², about 6.5cm², about 6.0 cm², about 5.5 cm², about 5.0 cm², or about 4.5 cm²;about 4.5 cm² to about 100 m², about 90 m², about 80 m², about 70 m²,about 60 m², about 50 m², about 45 m², about 40 m², about 35 m², about30 m², about 25 m², about 20 m², about 15 m², about 10 m², about 5 m²,about 1 m², about 800 cm², about 600 cm², about 400 cm², about 200 cm²,about 150 cm², about 100 cm², about 50 cm², about 48 cm², about 46 cm²,about 44 cm², about 42 cm², about 40 cm², about 38 cm², about 36 cm²,about 34 cm², about 32 cm², about 30 cm², about 28 cm², about 26 cm²,about 24 cm², about 22 cm², about 20 cm², about 18 cm², about 16 cm²,about 14 cm², about 12 cm², about 10 cm², about 9.5 cm², about 9.0 cm²,about 8.5 cm², about 8.0 cm², about 7.5 cm², about 7.0 cm², about 6.5cm², about 6.0 cm², about 5.5 cm², or about 5.0 cm²; about 5.0 cm² toabout 100 m², about 90 m², about 80 m², about 70 m², about 60 m², about50 m², about 45 m², about 40 m², about 35 m², about 30 m², about 25 m²,about 20 m², about 15 m², about 10 m², about 5 m², about 1 m², about 800cm², about 600 cm², about 400 cm², about 200 cm², about 150 cm², about100 cm², about 50 cm², about 48 cm², about 46 cm², about 44 cm², about42 cm², about 40 cm², about 38 cm², about 36 cm², about 34 cm², about 32cm², about 30 cm², about 28 cm², about 26 cm², about 24 cm², about 22cm², about 20 cm², about 18 cm², about 16 cm², about 14 cm², about 12cm², about 10 cm², about 9.5 cm², about 9.0 cm², about 8.5 cm², about8.0 cm², about 7.5 cm², about 7.0 cm², about 6.5 cm², about 6.0 cm², orabout 5.5 cm²; about 5.5 cm² to about 100 m², about 90 m², about 80 m²,about 70 m², about 60 m², about 50 m², about 45 m², about 40 m², about35 m², about 30 m², about 25 m², about 20 m², about 15 m², about 10 m²,about 5 m², about 1 m², about 800 cm², about 600 cm², about 400 cm²,about 200 cm², about 150 cm², about 100 cm², about 50 cm², about 48 cm²,about 46 cm², about 44 cm², about 42 cm², about 40 cm², about 38 cm²,about 36 cm², about 34 cm², about 32 cm², about 30 cm², about 28 cm²,about 26 cm², about 24 cm², about 22 cm², about 20 cm², about 18 cm²,about 16 cm², about 14 cm², about 12 cm², about 10 cm², about 9.5 cm²,about 9.0 cm², about 8.5 cm², about 8.0 cm², about 7.5 cm², about 7.0cm², about 6.5 cm², or about 6.0 cm²; about 6.0 cm² to about 100 m²,about 90 m², about 80 m², about 70 m², about 60 m², about 50 m², about45 m², about 40 m², about 35 m², about 30 m², about 25 m², about 20 m²,about 15 m², about 10 m², about 5 m², about 1 m², about 800 cm², about600 cm², about 400 cm², about 200 cm², about 150 cm², about 100 cm²,about 50 cm², about 48 cm², about 46 cm², about 44 cm², about 42 cm²,about 40 cm², about 38 cm², about 36 cm², about 34 cm², about 32 cm²,about 30 cm², about 28 cm², about 26 cm², about 24 cm², about 22 cm²,about 20 cm², about 18 cm², about 16 cm², about 14 cm², about 12 cm²,about 10 cm², about 9.5 cm², about 9.0 cm², about 8.5 cm², about 8.0cm², about 7.5 cm², about 7.0 cm², or about 6.5 cm²; about 6.5 cm² toabout 100 m², about 90 m², about 80 m², about 70 m², about 60 m², about50 m², about 45 m², about 40 m², about 35 m², about 30 m², about 25 m²,about 20 m², about 15 m², about 10 m², about 5 m², about 1 m², about 800cm², about 600 cm², about 400 cm², about 200 cm², about 150 cm², about100 cm², about 50 cm², about 48 cm², about 46 cm², about 44 cm², about42 cm², about 40 cm², about 38 cm², about 36 cm², about 34 cm², about 32cm², about 30 cm², about 28 cm², about 26 cm², about 24 cm², about 22cm², about 20 cm², about 18 cm², about 16 cm², about 14 cm², about 12cm², about 10 cm², about 9.5 cm², about 9.0 cm², about 8.5 cm², about8.0 cm², about 7.5 cm², or about 7.0 cm²; about 7.0 cm² to about 100 m²,about 90 m², about 80 m², about 70 m², about 60 m², about 50 m², about45 m², about 40 m², about 35 m², about 30 m², about 25 m², about 20 m²,about 15 m², about 10 m², about 5 m², about 1 m², about 800 cm², about600 cm², about 400 cm², about 200 cm², about 150 cm², about 100 cm²,about 50 cm², about 48 cm², about 46 cm², about 44 cm², about 42 cm²,about 40 cm², about 38 cm², about 36 cm², about 34 cm², about 32 cm²,about 30 cm², about 28 cm², about 26 cm², about 24 cm², about 22 cm²,about 20 cm², about 18 cm², about 16 cm², about 14 cm², about 12 cm²,about 10 cm², about 9.5 cm², about 9.0 cm², about 8.5 cm², about 8.0cm², or about 7.5 cm²; about 7.5 cm² to about 100 m², about 90 m², about80 m², about 70 m², about 60 m², about 50 m², about 45 m², about 40 m²,about 35 m², about 30 m², about 25 m², about 20 m², about 15 m², about10 m², about 5 m², about 1 m², about 800 cm², about 600 cm², about 400cm², about 200 cm², about 150 cm², about 100 cm², about 50 cm², about 48cm², about 46 cm², about 44 cm², about 42 cm², about 40 cm², about 38cm², about 36 cm², about 34 cm², about 32 cm², about 30 cm², about 28cm², about 26 cm², about 24 cm², about 22 cm², about 20 cm², about 18cm², about 16 cm², about 14 cm², about 12 cm², about 10 cm², about 9.5cm², about 9.0 cm², about 8.5 cm², or about 8.0 cm²; about 8.0 cm² toabout 100 m², about 90 m², about 80 m², about 70 m², about 60 m², about50 m², about 45 m², about 40 m², about 35 m², about 30 m², about 25 m²,about 20 m², about 15 m², about 10 m², about 5 m², about 1 m², about 800cm², about 600 cm², about 400 cm², about 200 cm², about 150 cm², about100 cm², about 50 cm², about 48 cm², about 46 cm², about 44 cm², about42 cm², about 40 cm², about 38 cm², about 36 cm², about 34 cm², about 32cm², about 30 cm², about 28 cm², about 26 cm², about 24 cm², about 22cm², about 20 cm², about 18 cm², about 16 cm², about 14 cm², about 12cm², about 10 cm², about 9.5 cm², about 9.0 cm², or about 8.5 cm²; about8.5 cm² to about 100 m², about 90 m², about 80 m², about 70 m², about 60m², about 50 m², about 45 m², about 40 m², about 35 m², about 30 m²,about 25 m², about 20 m², about 15 m², about 10 m², about 5 m², about 1m², about 800 cm², about 600 cm², about 400 cm², about 200 cm², about150 cm², about 100 cm², about 50 cm², about 48 cm², about 46 cm², about44 cm², about 42 cm², about 40 cm², about 38 cm², about 36 cm², about 34cm², about 32 cm², about 30 cm², about 28 cm², about 26 cm², about 24cm², about 22 cm², about 20 cm², about 18 cm², about 16 cm², about 14cm², about 12 cm², about 10 cm², about 9.5 cm², or about 9.0 cm²; about9.0 cm² to about 100 m², about 90 m², about 80 m², about 70 m², about 60m², about 50 m², about 45 m², about 40 m², about 35 m², about 30 m²,about 25 m², about 20 m², about 15 m², about 10 m², about 5 m², about 1m², about 800 cm², about 600 cm², about 400 cm², about 200 cm², about150 cm², about 100 cm², about 50 cm², about 48 cm², about 46 cm², about44 cm², about 42 cm², about 40 cm², about 38 cm², about 36 cm², about 34cm², about 32 cm², about 30 cm², about 28 cm², about 26 cm², about 24cm², about 22 cm², about 20 cm², about 18 cm², about 16 cm², about 14cm², about 12 cm², about 10 cm², or about 9.5 cm²; about 9.5 cm² toabout 100 m², about 90 m², about 80 m², about 70 m², about 60 m², about50 m², about 45 m², about 40 m², about 35 m², about 30 m², about 25 m²,about 20 m², about 15 m², about 10 m², about 5 m², about 1 m², about 800cm², about 600 cm², about 400 cm², about 200 cm², about 150 cm², about100 cm², about 50 cm², about 48 cm², about 46 cm², about 44 cm², about42 cm², about 40 cm², about 38 cm², about 36 cm², about 34 cm², about 32cm², about 30 cm², about 28 cm², about 26 cm², about 24 cm², about 22cm², about 20 cm², about 18 cm², about 16 cm², about 14 cm², about 12cm², or about 10 cm²; about 10 cm² to about 100 m², about 90 m², about80 m², about 70 m², about 60 m², about 50 m², about 45 m², about 40 m²,about 35 m², about 30 m², about 25 m², about 20 m², about 15 m², about10 m², about 5 m², about 1 m², about 800 cm², about 600 cm², about 400cm², about 200 cm², about 150 cm², about 100 cm², about 50 cm², about 48cm², about 46 cm², about 44 cm², about 42 cm², about 40 cm², about 38cm², about 36 cm², about 34 cm², about 32 cm², about 30 cm², about 28cm², about 26 cm², about 24 cm², about 22 cm², about 20 cm², about 18cm², about 16 cm², about 14 cm², or about 12 cm²; about 12 cm² to about100 m², about 90 m², about 80 m², about 70 m², about 60 m², about 50 m²,about 45 m², about 40 m², about 35 m², about 30 m², about 25 m², about20 m², about 15 m², about 10 m², about 5 m², about 1 m², about 800 cm²,about 600 cm², about 400 cm², about 200 cm², about 150 cm², about 100cm², about 50 cm², about 48 cm², about 46 cm², about 44 cm², about 42cm², about 40 cm², about 38 cm², about 36 cm², about 34 cm², about 32cm², about 30 cm², about 28 cm², about 26 cm², about 24 cm², about 22cm², about 20 cm², about 18 cm², about 16 cm², or about 14 cm²; about 14cm² to about 100 m², about 90 m², about 80 m², about 70 m², about 60 m²,about 50 m², about 45 m², about 40 m², about 35 m², about 30 m², about25 m², about 20 m², about 15 m², about 10 m², about 5 m², about 1 m²,about 800 cm², about 600 cm², about 400 cm², about 200 cm², about 150cm², about 100 cm², about 50 cm², about 48 cm², about 46 cm², about 44cm², about 42 cm², about 40 cm², about 38 cm², about 36 cm², about 34cm², about 32 cm², about 30 cm², about 28 cm², about 26 cm², about 24cm², about 22 cm², about 20 cm², about 18 cm², or about 16 cm²; about 16cm² to about 100 m², about 90 m², about 80 m², about 70 m², about 60 m²,about 50 m², about 45 m², about 40 m², about 35 m², about 30 m², about25 m², about 20 m², about 15 m², about 10 m², about 5 m², about 1 m²,about 800 cm², about 600 cm², about 400 cm², about 200 cm², about 150cm², about 100 cm², about 50 cm², about 48 cm², about 46 cm², about 44cm², about 42 cm², about 40 cm², about 38 cm², about 36 cm², about 34cm², about 32 cm², about 30 cm², about 28 cm², about 26 cm², about 24cm², about 22 cm², about 20 cm², or about 18 cm²; about 18 cm² to about100 m², about 90 m², about 80 m², about 70 m², about 60 m², about 50 m²,about 45 m², about 40 m², about 35 m², about 30 m², about 25 m², about20 m², about 15 m², about 10 m², about 5 m², about 1 m², about 800 cm²,about 600 cm², about 400 cm², about 200 cm², about 150 cm², about 100cm², about 50 cm², about 48 cm², about 46 cm², about 44 cm², about 42cm², about 40 cm², about 38 cm², about 36 cm², about 34 cm², about 32cm², about 30 cm², about 28 cm², about 26 cm², about 24 cm², about 22cm², or about 20 cm²; about 20 cm² to about 100 m², about 90 m², about80 m², about 70 m², about 60 m², about 50 m², about 45 m², about 40 m²,about 35 m², about 30 m², about 25 m², about 20 m², about 15 m², about10 m², about 5 m², about 1 m², about 800 cm², about 600 cm², about 400cm², about 200 cm², about 150 cm², about 100 cm², about 50 cm², about 48cm², about 46 cm², about 44 cm², about 42 cm², about 40 cm², about 38cm², about 36 cm², about 34 cm², about 32 cm², about 30 cm², about 28cm², about 26 cm², about 24 cm², or about 22 cm²; about 22 cm² to about100 m², about 90 m², about 80 m², about 70 m², about 60 m², about 50 m²,about 45 m², about 40 m², about 35 m², about 30 m², about 25 m², about20 m², about 15 m², about 10 m², about 5 m², about 1 m², about 800 cm²,about 600 cm², about 400 cm², about 200 cm², about 150 cm², about 100cm², about 50 cm², about 48 cm², about 46 cm², about 44 cm², about 42cm², about 40 cm², about 38 cm², about 36 cm², about 34 cm², about 32cm², about 30 cm², about 28 cm², about 26 cm², or about 24 cm²; about 24cm² to about 100 m², about 90 m², about 80 m², about 70 m², about 60 m²,about 50 m², about 45 m², about 40 m², about 35 m², about 30 m², about25 m², about 20 m², about 15 m², about 10 m², about 5 m², about 1 m²,about 800 cm², about 600 cm², about 400 cm², about 200 cm², about 150cm², about 100 cm², about 50 cm², about 48 cm², about 46 cm², about 44cm², about 42 cm², about 40 cm², about 38 cm², about 36 cm², about 34cm², about 32 cm², about 30 cm², about 28 cm², or about 26 cm²; about 26cm² to about 100 m², about 90 m², about 80 m², about 70 m², about 60 m²,about 50 m², about 45 m², about 40 m², about 35 m², about 30 m², about25 m², about 20 m², about 15 m², about 10 m², about 5 m², about 1 m²,about 800 cm², about 600 cm², about 400 cm², about 200 cm², about 150cm², about 100 cm², about 50 cm², about 48 cm², about 46 cm², about 44cm², about 42 cm², about 40 cm², about 38 cm², about 36 cm², about 34cm², about 32 cm², about 30 cm², or about 28 cm²; about 28 cm² to about100 m², about 90 m², about 80 m², about 70 m², about 60 m², about 50 m²,about 45 m², about 40 m², about 35 m², about 30 m², about 25 m², about20 m², about 15 m², about 10 m², about 5 m², about 1 m², about 800 cm²,about 600 cm², about 400 cm², about 200 cm², about 150 cm², about 100cm², about 50 cm², about 48 cm², about 46 cm², about 44 cm², about 42cm², about 40 cm², about 38 cm², about 36 cm², about 34 cm², about 32cm², or about 30 cm²; about 30 cm² to about 100 m², about 90 m², about80 m², about 70 m², about 60 m², about 50 m², about 45 m², about 40 m²,about 35 m², about 30 m², about 25 m², about 20 m², about 15 m², about10 m², about 5 m², about 1 m², about 800 cm², about 600 cm², about 400cm², about 200 cm², about 150 cm², about 100 cm², about 50 cm², about 48cm², about 46 cm², about 44 cm², about 42 cm², about 40 cm², about 38cm², about 36 cm², about 34 cm², or about 32 cm²; about 32 cm² to about100 m², about 90 m², about 80 m², about 70 m², about 60 m², about 50 m²,about 45 m², about 40 m², about 35 m², about 30 m², about 25 m², about20 m², about 15 m², about 10 m², about 5 m², about 1 m², about 800 cm²,about 600 cm², about 400 cm², about 200 cm², about 150 cm², about 100cm², about 50 cm², about 48 cm², about 46 cm², about 44 cm², about 42cm², about 40 cm², about 38 cm², about 36 cm², or about 34 cm²; about 34cm² to about 100 m², about 90 m², about 80 m², about 70 m², about 60 m²,about 50 m², about 45 m², about 40 m², about 35 m², about 30 m², about25 m², about 20 m², about 15 m², about 10 m², about 5 m², about 1 m²,about 800 cm², about 600 cm², about 400 cm², about 200 cm², about 150cm², about 100 cm², about 50 cm², about 48 cm², about 46 cm², about 44cm², about 42 cm², about 40 cm², about 38 cm², or about 36 cm²; about 36cm² to about 100 m², about 90 m², about 80 m², about 70 m², about 60 m²,about 50 m², about 45 m², about 40 m², about 35 m², about 30 m², about25 m², about 20 m², about 15 m², about 10 m², about 5 m², about 1 m²,about 800 cm², about 600 cm², about 400 cm², about 200 cm², about 150cm², about 100 cm², about 50 cm², about 48 cm², about 46 cm², about 44cm², about 42 cm², about 40 cm², or about 38 cm²; about 38 cm² to about100 m², about 90 m², about 80 m², about 70 m², about 60 m², about 50 m²,about 45 m², about 40 m², about 35 m², about 30 m², about 25 m², about20 m², about 15 m², about 10 m², about 5 m², about 1 m², about 800 cm²,about 600 cm², about 400 cm², about 200 cm², about 150 cm², about 100cm², about 50 cm², about 48 cm², about 46 cm², about 44 cm², about 42cm², or about 40 cm²; about 40 cm² to about 100 m², about 90 m², about80 m², about 70 m², about 60 m², about 50 m², about 45 m², about 40 m²,about 35 m², about 30 m², about 25 m², about 20 m², about 15 m², about10 m², about 5 m², about 1 m², about 800 cm², about 600 cm², about 400cm², about 200 cm², about 150 cm², about 100 cm², about 50 cm², about 48cm², about 46 cm², about 44 cm², or about 42 cm²; about 42 cm² to about100 m², about 90 m², about 80 m², about 70 m², about 60 m², about 50 m²,about 45 m², about 40 m², about 35 m², about 30 m², about 25 m², about20 m², about 15 m², about 10 m², about 5 m², about 1 m², about 800 cm²,about 600 cm², about 400 cm², about 200 cm², about 150 cm², about 100cm², about 50 cm², about 48 cm², about 46 cm², or about 44 cm²; about 44cm² to about 100 m², about 90 m², about 80 m², about 70 m², about 60 m²,about 50 m², about 45 m², about 40 m², about 35 m², about 30 m², about25 m², about 20 m², about 15 m², about 10 m², about 5 m², about 1 m²,about 800 cm², about 600 cm², about 400 cm², about 200 cm², about 150cm², about 100 cm², about 50 cm², about 48 cm², or about 46 cm²; about46 cm² to about 100 m², about 90 m², about 80 m², about 70 m², about 60m², about 50 m², about 45 m², about 40 m², about 35 m², about 30 m²,about 25 m², about 20 m², about 15 m², about 10 m², about 5 m², about 1m², about 800 cm², about 600 cm², about 400 cm², about 200 cm², about150 cm², about 100 cm², about 50 cm² or about 48 cm²; about 48 cm² toabout 100 m², about 90 m², about 80 m², about 70 m², about 60 m², about50 m², about 45 m², about 40 m², about 35 m², about 30 m², about 25 m²,about 20 m², about 15 m², about 10 m², about 5 m², about 1 m², about 800cm², about 600 cm², about 400 cm², about 200 cm², about 150 cm², about100 cm², or about 50 cm²; about 50 cm² to about 100 m², about 90 m²,about 80 m², about 70 m², about 60 m², about 50 m², about 45 m², about40 m², about 35 m², about 30 m², about 25 m², about 20 m², about 15 m²,about 10 m², about 5 m², about 1 m², about 800 cm², about 600 cm², about400 cm², about 200 cm², about 150 cm², or about 100 cm²; about 100 cm²to about 100 m², about 90 m², about 80 m², about 70 m², about 60 m²,about 50 m², about 45 m², about 40 m², about 35 m², about 30 m², about25 m², about 20 m², about 15 m², about 10 m², about 5 m², about 1 m²,about 800 cm², about 600 cm², about 400 cm², about 200 cm², or about 150cm²; about 150 cm² to about 100 m², about 90 m², about 80 m², about 70m², about 60 m², about 100 m², about 90 m², about 80 m², about 70 m²,about 60 m², about 50 m², about 45 m², about 40 m², about 35 m², about30 m², about 25 m², about 20 m², about 15 m², about 10 m², about 5 m²,about 1 m², about 800 cm², about 600 cm², about 400 cm², or about 200cm²; about 200 cm² to about 100 m², about 90 m², about 80 m², about 70m², about 60 m², about 50 m², about 45 m², about 40 m², about 35 m²,about 30 m², about 25 m², about 20 m², about 15 m², about 10 m², about 5m², about 1 m², about 800 cm², about 600 cm², or about 400 cm²; about400 cm² to about 100 m², about 90 m², about 80 m², about 70 m², about 60m², about 50 m², about 45 m², about 40 m², about 35 m², about 30 m²,about 25 m², about 20 m², about 15 m², about 10 m², about 5 m², about 1m², about 800 cm², or about 600 cm²; about 600 cm² to about 100 m²,about 90 m², about 80 m², about 70 m², about 60 m², about 50 m², about45 m², about 40 m², about 35 m², about 30 m², about 25 m², about 20 m²,about 15 m², about 10 m², about 5 m², about 1 m², about 800 cm²; about800 cm² to about 100 m², about 90 m², about 80 m², about 70 m², about 60m², about 50 m², about 45 m², about 40 m², about 35 m², about 30 m²,about 25 m², about 20 m², about 15 m², about 10 m², about 5 m², or about1 m²; about 1 m² to about 50 m², about 45 m², about 40 m², about 35 m²,about 30 m², about 25 m², about 20 m², about 15 m², about 10 m², orabout 5 m²; about 5 m² to about 100 m², about 90 m², about 80 m², about70 m², about 60 m², about 50 m², about 45 m², about 40 m², about 35 m²,about 30 m², about 25 m², about 20 m², about 15 m², or about 10 m²;about 10 m² to about 100 m², about 90 m², about 80 m², about 70 m²,about 60 m², about 50 m², about 45 m², about 40 m², about 35 m², about30 m², about 25 m², about 20 m², or about 15 m²; about 15 m² to about100 m², about 90 m², about 80 m², about 70 m², about 60 m², about 50 m²,about 45 m², about 40 m², about 35 m², about 30 m², about 25 m², orabout 20 m²; about 20 m² to about 100 m², about 90 m², about 80 m²,about 70 m², about 60 m², about 50 m², about 45 m², about 40 m², about35 m², about 30 m², or about 25 m²; about 25 m² to about 100 m², about90 m², about 80 m², about 70 m², about 60 m², about 50 m², about 45 m²,about 40 m², about 35 m², or about 30 m²; about 30 m² to about 50 m²,about 45 m², about 40 m², or about 35 m²; about 35 m² to about 100 m²,about 90 m², about 80 m², about 70 m², about 60 m², about 50 m², about45 m², or about 40 m²; about 40 m² to about 100 m², about 90 m², about80 m², about 70 m², about 60 m², about 50 m² or about 45 m²; about 45 m²to about 100 m², about 90 m², about 80 m², about 70 m², about 60 m², orabout 50 m²; about 50 m² to about 100 m², about 90 m², about 80 m²,about 70 m², or about 60 m²; about 60 m² to about 100 m², about 90 m²,about 80 m², or about 70 m²; about 70 m² to about 100 m², about 90 m²,or about 80 m²; about 80 m² to about 100 m², or about 90 m²; or about 90m² to about 100 m².

In some embodiments, the TFVF unit includes one or more tangential virusfilters having a pore size of about 5 nm to about 220 nm, about 210 nm,about 200 nm, about 190 nm, about 180 nm, about 170 nm, about 160 nm,about 150 nm, about 140 nm, about 130 nm, about 120 nm, about 110 nm,about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, about 50 nm,about 45 nm, about 40 nm, about 35 nm, about 30 nm, about 25 nm, about20 nm, about 15 nm, or about 10 nm; about 10 nm to about 220 nm, about210 nm, about 200 nm, about 190 nm, about 180 nm, about 170 nm, about160 nm, about 150 nm, about 140 nm, about 130 nm, about 120 nm, about110 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm,about 50 nm, about 45 nm, about 40 nm, about 35 nm, about 30 nm, about25 nm, about 20 nm, or about 15 nm; about 15 nm to about 220 nm, about210 nm, about 200 nm, about 190 nm, about 180 nm, about 170 nm, about160 nm, about 150 nm, about 140 nm, about 130 nm, about 120 nm, about110 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm,about 50 nm, about 45 nm, about 40 nm, about 35 nm, about 30 nm, about25 nm, or about 20 nm; about 20 nm to about 220 nm, about 210 nm, about200 nm, about 190 nm, about 180 nm, about 170 nm, about 160 nm, about150 nm, about 140 nm, about 130 nm, about 120 nm, about 110 nm, about100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm,about 70 nm, about 65 nm, about 60 nm, about 55 nm, about 50 nm, about45 nm, about 40 nm, about 35 nm, about 30 nm, or about 25 nm; about 25nm to about 220 nm, about 210 nm, about 200 nm, about 190 nm, about 180nm, about 170 nm, about 160 nm, about 150 nm, about 140 nm, about 130nm, about 120 nm, about 110 nm, about 100 nm, about 95 nm, about 90 nm,about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about60 nm, about 55 nm, about 50 nm, about 45 nm, about 40 nm, about 35 nm,or about 30 nm; about 30 nm to about 220 nm, about 210 nm, about 200 nm,about 190 nm, about 180 nm, about 170 nm, about 160 nm, about 150 nm,about 140 nm, about 130 nm, about 120 nm, about 110 nm, about 100 nm,about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about70 nm, about 65 nm, about 60 nm, about 55 nm, about 50 nm, about 45 nm,about 40 nm, or about 35 nm; about 35 nm to about 220 nm, about 210 nm,about 200 nm, about 190 nm, about 180 nm, about 170 nm, about 160 nm,about 150 nm, about 140 nm, about 130 nm, about 120 nm, about 110 nm,about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, about 50 nm,about 45 nm, or about 40 nm; about 45 nm to about 220 nm, about 210 nm,about 200 nm, about 190 nm, about 180 nm, about 170 nm, about 160 nm,about 150 nm, about 140 nm, about 130 nm, about 120 nm, about 110 nm,about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50nm; about 50 nm to about 220 nm, about 210 nm, about 200 nm, about 190nm, about 180 nm, about 170 nm, about 160 nm, about 150 nm, about 140nm, about 130 nm, about 120 nm, about 110 nm, about 100 nm, about 95 nm,about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about65 nm, about 60 nm, or about 55 nm; about 55 nm to about 220 nm, about210 nm, about 200 nm, about 190 nm, about 180 nm, about 170 nm, about160 nm, about 150 nm, about 140 nm, about 130 nm, about 120 nm, about110 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80nm, about 75 nm, about 70 nm, about 65 nm, or about 60 nm; about 60 nmto about 220 nm, about 210 nm, about 200 nm, about 190 nm, about 180 nm,about 170 nm, about 160 nm, about 150 nm, about 140 nm, about 130 nm,about 120 nm, about 110 nm, about 100 nm, about 95 nm, about 90 nm,about 85 nm, about 80 nm, about 75 nm, about 70 nm, or about 65 nm;about 65 nm to about 220 nm, about 210 nm, about 200 nm, about 190 nm,about 180 nm, about 170 nm, about 160 nm, about 150 nm, about 140 nm,about 130 nm, about 120 nm, about 110 nm, about 100 nm, about 95 nm,about 90 nm, about 85 nm, about 80 nm, about 75 nm, or about 70 nm;about 70 nm to about 220 nm, about 210 nm, about 200 nm, about 190 nm,about 180 nm, about 170 nm, about 160 nm, about 150 nm, about 140 nm,about 130 nm, about 120 nm, about 110 nm, about 100 nm, about 95 nm,about 90 nm, about 85 nm, about 80 nm, or about 75 nm; about 75 nm toabout 220 nm, about 210 nm, about 200 nm, about 190 nm, about 180 nm,about 170 nm, about 160 nm, about 150 nm, about 140 nm, about 130 nm,about 120 nm, about 110 nm, about 100 nm, about 95 nm, about 90 nm,about 85 nm, or about 80 nm; about 80 nm to about 220 nm, about 210 nm,about 200 nm, about 190 nm, about 180 nm, about 170 nm, about 160 nm,about 150 nm, about 140 nm, about 130 nm, about 120 nm, about 110 nm,about 100 nm, about 95 nm, about 90 nm, or about 85 nm; about 85 nm toabout 220 nm, about 210 nm, about 200 nm, about 190 nm, about 180 nm,about 170 nm, about 160 nm, about 150 nm, about 140 nm, about 130 nm,about 120 nm, about 110 nm, about 100 nm, about 95 nm, or about 90 nm;about 90 nm to about 220 nm, about 210 nm, about 200 nm, about 190 nm,about 180 nm, about 170 nm, about 160 nm, about 150 nm, about 140 nm,about 130 nm, about 120 nm, about 110 nm, about 100 nm, or about 95 nm;about 95 nm to about 220 nm, about 210 nm, about 200 nm, about 190 nm,about 180 nm, about 170 nm, about 160 nm, about 150 nm, about 140 nm,about 130 nm, about 120 nm, about 110 nm, or about 100 nm; about 100 nmto about 220 nm, about 210 nm, about 200 nm, about 190 nm, about 180 nm,about 170 nm, about 160 nm, about 150 nm, about 140 nm, about 130 nm,about 120 nm, or about 110 nm; about 110 nm to about 220 nm, about 210nm, about 200 nm, about 190 nm, about 180 nm, about 170 nm, about 160nm, about 150 nm, about 140 nm, about 130 nm, or about 120 nm; about 120nm to about 220 nm, about 210 nm, about 200 nm, about 190 nm, about 180nm, about 170 nm, about 160 nm, about 150 nm, about 140 nm, or about 130nm; about 130 nm to about 220 nm, about 210 nm, about 200 nm, about 190nm, about 180 nm, about 170 nm, about 160 nm, about 150 nm, or about 140nm; about 140 nm to about 220 nm, about 210 nm, about 200 nm, about 190nm, about 180 nm, about 170 nm, about 160 nm, or about 150 nm; about 150nm to about 220 nm, about 210 nm, about 200 nm, about 190 nm, about 180nm, about 170 nm, or about 160 nm; about 160 nm to about 220 nm, about210 nm, about 200 nm, about 190 nm, about 180 nm, or about 170 nm; about170 nm to about 220 nm, about 210 nm, about 200 nm, about 190 nm, orabout 180 nm; about 180 nm to about 220 nm, about 210 nm, about 200 nm,or about 190 nm; about 190 nm to about 220 nm, about 210 nm, or about200 nm; about 200 nm to about 220 nm or about 210 nm; or about 210 nm toabout 220 nm. A variety of virus filters that can be used in a TFVF unitare known in the art. For example, virus filters that can be used in aTFVF unit are commercially available from Asahi Kasei (Tokyo, Japan). Insome embodiments, the virus filter is a Planova™ filter (e.g, a Planova™15N, a Planova™ 20N, a Planova™ 35N, or a Planova™ 75N). In someembodiments, the virus filter is a Planova™ BioEX filter. The virusfilters that can be used in a TFVF unit can include, e.g., hollow fibermembranes.

The connecting conduit and the conduit in the second circuit system canbe simple tubing, e.g., biocompatible tubing. Non-limiting examples ofuseful tubing include silicone rubber, polycarbonate, polypropylene,polyurethane, polydioxanone (PDO), polyhydroxyalkanoate,polyhydroxybutyrate, poly(glycerol sebacate), polyglycolide,polylactide, polycaprolactone, or polyanhydride, or copolymers orderivatives including these and/or other polymers. Alternatively or inaddition, any of the conduits described herein can include polyvinylchloride. Any of the conduits described herein can be, e.g., weldabletransfer tubing.

In some embodiments, the internal diameter of the connecting conduitand/or the conduit in the second circuit system can be about 5 mm toabout 100 mm, about 95 mm, about 90 mm, about 85 mm, about 80 mm, about75 mm, about 70 mm, about 65 mm, about 60 mm, about 55 mm, about 50 mm,about 48 mm, about 46 mm, about 44 mm, about 42 mm, about 40 mm, about38 mm, about 36 mm, about 34 mm, about 32 mm, about 30 mm, about 28 mm,about 26 mm, about 24 mm, about 22 mm, about 20 mm, about 18 mm, about16 mm, about 14 mm, about 12 mm, about 10 mm, about 8 mm, or about 6 mm;about 6 mm to about 100 mm, about 95 mm, about 90 mm, about 85 mm, about80 mm, about 75 mm, about 70 mm, about 65 mm, about 60 mm, about 55 mm,about 50 mm, about 48 mm, about 46 mm, about 44 mm, about 42 mm, about40 mm, about 38 mm, about 36 mm, about 34 mm, about 32 mm, about 30 mm,about 28 mm, about 26 mm, about 24 mm, about 22 mm, about 20 mm, about18 mm, about 16 mm, about 14 mm, about 12 mm, about 10 mm, or about 8mm; about 8 mM to about 100 mm, about 95 mm, about 90 mm, about 85 mm,about 80 mm, about 75 mm, about 70 mm, about 65 mm, about 60 mm, about55 mm, about 50 mm, about 48 mm, about 46 mm, about 44 mm, about 42 mm,about 40 mm, about 38 mm, about 36 mm, about 34 mm, about 32 mm, about30 mm, about 28 mm, about 26 mm, about 24 mm, about 22 mm, about 20 mm,about 18 mm, about 16 mm, about 14 mm, about 12 mm, or about 10 mm;about 10 mm to about 100 mm, about 95 mm, about 90 mm, about 85 mm,about 80 mm, about 75 mm, about 70 mm, about 65 mm, about 60 mm, about55 mm, about 50 mm, about 48 mm, about 46 mm, about 44 mm, about 42 mm,about 40 mm, about 38 mm, about 36 mm, about 34 mm, about 32 mm, about30 mm, about 28 mm, about 26 mm, about 24 mm, about 22 mm, about 20 mm,about 18 mm, about 16 mm, about 14 mm, or about 12 mm; about 12 mm toabout 100 mm, about 95 mm, about 90 mm, about 85 mm, about 80 mm, about75 mm, about 70 mm, about 65 mm, about 60 mm, about 55 mm, about 50 mm,about 48 mm, about 46 mm, about 44 mm, about 42 mm, about 40 mm, about38 mm, about 36 mm, about 34 mm, about 32 mm, about 30 mm, about 28 mm,about 26 mm, about 24 mm, about 22 mm, about 20 mm, about 18 mm, about16 mm, or about 14 mm; about 14 mm to about 100 mm, about 95 mm, about90 mm, about 85 mm, about 80 mm, about 75 mm, about 70 mm, about 65 mm,about 60 mm, about 55 mm, about 50 mm, about 48 mm, about 46 mm, about44 mm, about 42 mm, about 40 mm, about 38 mm, about 36 mm, about 34 mm,about 32 mm, about 30 mm, about 28 mm, about 26 mm, about 24 mm, about22 mm, about 20 mm, about 18 mm, or about 16 mm; about 16 mm to about100 mm, about 95 mm, about 90 mm, about 85 mm, about 80 mm, about 75 mm,about 70 mm, about 65 mm, about 60 mm, about 55 mm, about 50 mm, about48 mm, about 46 mm, about 44 mm, about 42 mm, about 40 mm, about 38 mm,about 36 mm, about 34 mm, about 32 mm, about 30 mm, about 28 mm, about26 mm, about 24 mm, about 22 mm, about 20 mm, or about 18 mm; about 18mm to about 100 mm, about 95 mm, about 90 mm, about 85 mm, about 80 mm,about 75 mm, about 70 mm, about 65 mm, about 60 mm, about 55 mm, about50 mm, about 48 mm, about 46 mm, about 44 mm, about 42 mm, about 40 mm,about 38 mm, about 36 mm, about 34 mm, about 32 mm, about 30 mm, about28 mm, about 26 mm, about 24 mm, about 22 mm, or about 20 mm; about 20mm to about 100 mm, about 95 mm, about 90 mm, about 85 mm, about 80 mm,about 75 mm, about 70 mm, about 65 mm, about 60 mm, about 55 mm, about50 mm, about 48 mm, about 46 mm, about 44 mm, about 42 mm, about 40 mm,about 38 mm, about 36 mm, about 34 mm, about 32 mm, about 30 mm, about28 mm, about 26 mm, about 24 mm, or about 22 mm; about 22 mm to about100 mm, about 95 mm, about 90 mm, about 85 mm, about 80 mm, about 75 mm,about 70 mm, about 65 mm, about 60 mm, about 55 mm, about 50 mm, about48 mm, about 46 mm, about 44 mm, about 42 mm, about 40 mm, about 38 mm,about 36 mm, about 34 mm, about 32 mm, about 30 mm, about 28 mm, about26 mm, or about 24 mm; about 24 mm to about 100 mm, about 95 mm, about90 mm, about 85 mm, about 80 mm, about 75 mm, about 70 mm, about 65 mm,about 60 mm, about 55 mm, about 50 mm, about 48 mm, about 46 mm, about44 mm, about 42 mm, about 40 mm, about 38 mm, about 36 mm, about 34 mm,about 32 mm, about 30 mm, about 28 mm, or about 26 mm; about 26 mm toabout 50 mm, about 48 mm, about 46 mm, about 44 mm, about 42 mm, about40 mm, about 38 mm, about 36 mm, about 34 mm, about 32 mm, about 30 mm,or about 28 mm; about 28 mm to about 100 mm, about 95 mm, about 90 mm,about 85 mm, about 80 mm, about 75 mm, about 70 mm, about 65 mm, about60 mm, about 55 mm, about 50 mm, about 48 mm, about 46 mm, about 44 mm,about 42 mm, about 40 mm, about 38 mm, about 36 mm, about 34 mm, about32 mm, or about 30 mm; about 30 mm to about 100 mm, about 95 mm, about90 mm, about 85 mm, about 80 mm, about 75 mm, about 70 mm, about 65 mm,about 60 mm, about 55 mm, about 50 mm, about 48 mm, about 46 mm, about44 mm, about 42 mm, about 40 mm, about 38 mm, about 36 mm, about 34 mm,or about 32 mm; about 32 mm to about 100 mm, about 95 mm, about 90 mm,about 85 mm, about 80 mm, about 75 mm, about 70 mm, about 65 mm, about60 mm, about 55 mm, about 50 mm, about 48 mm, about 46 mm, about 44 mm,about 42 mm, about 40 mm, about 38 mm, about 36 mm, or about 34 mm;about 34 mm to about 100 mm, about 95 mm, about 90 mm, about 85 mm,about 80 mm, about 75 mm, about 70 mm, about 65 mm, about 60 mm, about55 mm, about 50 mm, about 48 mm, about 46 mm, about 44 mm, about 42 mm,about 40 mm, about 38 mm, or about 36 mm; about 36 mm to about 100 mm,about 95 mm, about 90 mm, about 85 mm, about 80 mm, about 75 mm, about70 mm, about 65 mm, about 60 mm, about 55 mm, about 50 mm, about 48 mm,about 46 mm, about 44 mm, about 42 mm, about 40 mm, to about 38 mm;about 38 mm to about 100 mm, about 95 mm, about 90 mm, about 85 mm,about 80 mm, about 75 mm, about 70 mm, about 65 mm, about 60 mm, about55 mm, about 50 mm, about 48 mm, about 46 mm, about 44 mm, about 42 mm,or about 40 mm; about 40 mm to about 100 mm, about 95 mm, about 90 mm,about 85 mm, about 80 mm, about 75 mm, about 70 mm, about 65 mm, about60 mm, about 55 mm, about 50 mm, about 48 mm, about 46 mm, about 44 mm,or about 42 mm; about 42 mm to about 100 mm, about 95 mm, about 90 mm,about 85 mm, about 80 mm, about 75 mm, about 70 mm, about 65 mm, about60 mm, about 55 mm, about 50 mm, about 48 mm, about 46 mm, or about 44mm; about 44 mm to about 100 mm, about 95 mm, about 90 mm, about 85 mm,about 80 mm, about 75 mm, about 70 mm, about 65 mm, about 60 mm, about55 mm, about 50 mm, about 48 mm, or about 46 mm; about 46 mm to about100 mm, about 95 mm, about 90 mm, about 85 mm, about 80 mm, about 75 mm,about 70 mm, about 65 mm, about 60 mm, about 55 mm, about 50 mm, orabout 48 mm; about 48 mm to about 100 mm, about 95 mm, about 90 mm,about 85 mm, about 80 mm, about 75 mm, about 70 mm, about 65 mm, about60 mm, about 55 mm, or about 50 mm; about 50 mm to about 100 mm, about95 mm, about 90 mm, about 85 mm, about 80 mm, about 75 mm, about 70 mm,about 65 mm, about 60 mm, or about 55 mm; about 55 mm to about 100 mm,about 95 mm, about 90 mm, about 85 mm, about 80 mm, about 75 mm, about70 mm, about 65 mm, or about 60 mm; about 60 mm to about 100 mm, about95 mm, about 90 mm, about 85 mm, about 80 mm, about 75 mm, about 70 mm,or about 65 mm; about 65 mm to about 100 mm, about 95 mm, about 90 mm,about 85 mm, about 80 mm, about 75 mm, or about 70 mm; about 70 mm toabout 100 mm, about 95 mm, about 90 mm, about 85 mm, about 80 mm, orabout 75 mm; about 75 mm to about 100 mm, about 95 mm, about 90 mm,about 85 mm, or about 80 mm; about 80 mm to about 100 mm, about 95 mm,about 90 mm, or about 85 mm; about 85 mm to about 100 mm, about 95 mm,or about 90 mm; about 90 mm to about 100 mm or about 95 mm; or about 95mm to about 100 mm.

In some embodiments, the connecting conduit and/or the conduit in thesecond circuit system has a total length of about 1.0 cm to about 75 cm,about 70 cm, about 65 cm, about 60 cm, about 55 cm, about 50 cm, about45 cm, about 40 cm, about 35 cm, about 30 cm, about 25 cm, about 20 cm,about 18 cm, about 16 cm, about 14 cm, about 12 cm, about 10 cm, about8.0 cm, about 6.0 cm, about 4.0 cm, or about 2.0 cm; about 2.0 cm toabout 75 cm, about 70 cm, about 65 cm, about 60 cm, about 55 cm, about50 cm, about 45 cm, about 40 cm, about 35 cm, about 30 cm, about 25 cm,about 20 cm, about 18 cm, about 16 cm, about 14 cm, about 12 cm, about10 cm, about 8.0 cm, about 6.0 cm, or about 4.0 cm; about 4.0 cm toabout 75 cm, about 70 cm, about 65 cm, about 60 cm, about 55 cm, about50 cm, about 45 cm, about 40 cm, about 35 cm, about 30 cm, about 25 cm,about 20 cm, about 18 cm, about 16 cm, about 14 cm, about 12 cm, about10 cm, about 8.0 cm, or about 6.0 cm; about 6.0 cm to about 75 cm, about70 cm, about 65 cm, about 60 cm, about 55 cm, about 50 cm, about 45 cm,about 40 cm, about 35 cm, about 30 cm, about 25 cm, about 20 cm, about18 cm, about 16 cm, about 14 cm, about 12 cm, about 10 cm, or about 8.0cm; about 8.0 cm to about 75 cm, about 70 cm, about 65 cm, about 60 cm,about 55 cm, about 50 cm, about 45 cm, about 40 cm, about 35 cm, about30 cm, about 25 cm, about 20 cm, about 18 cm, about 16 cm, about 14 cm,about 12 cm, or about 10 cm; about 10 cm to about 75 cm, about 70 cm,about 65 cm, about 60 cm, about 55 cm, about 50 cm, about 45 cm, about40 cm, about 35 cm, about 30 cm, about 25 cm, about 20 cm, about 18 cm,about 16 cm, about 14 cm, or about 12 cm; about 12 cm to about 75 cm,about 70 cm, about 65 cm, about 60 cm, about 55 cm, about 50 cm, about45 cm, about 40 cm, about 35 cm, about 30 cm, about 25 cm, about 20 cm,about 18 cm, about 16 cm, or about 14 cm; about 14 cm to about 75 cm,about 70 cm, about 65 cm, about 60 cm, about 55 cm, about 50 cm, about45 cm, about 40 cm, about 35 cm, about 30 cm, about 25 cm, about 20 cm,about 18 cm, or about 16 cm; about 16 cm to about 75 cm, about 70 cm,about 65 cm, about 60 cm, about 55 cm, about 50 cm, about 45 cm, about40 cm, about 35 cm, about 30 cm, about 25 cm, about 20 cm, or about 18cm; about 18 cm to about 75 cm, about 70 cm, about 65 cm, about 60 cm,about 55 cm, about 50 cm, about 45 cm, about 40 cm, about 35 cm, about30 cm, about 25 cm, or about 20 cm; about 20 cm to about 75 cm, about 70cm, about 65 cm, about 60 cm, about 55 cm, about 50 cm, about 45 cm,about 40 cm, about 35 cm, about 30 cm, or about 25 cm; about 25 cm toabout 75 cm, about 70 cm, about 65 cm, about 60 cm, about 55 cm, about50 cm, about 45 cm, about 40 cm, about 35 cm, or about 30 cm; about 30cm to about 75 cm, about 70 cm, about 65 cm, about 60 cm, about 55 cm,about 50 cm, about 45 cm, about 40 cm, or about 35 cm; about 35 cm toabout 75 cm, about 70 cm, about 65 cm, about 60 cm, about 55 cm, about50 cm, about 45 cm, or about 40 cm; about 40 cm to about 75 cm, about 70cm, about 65 cm, about 60 cm, about 55 cm, about 50 cm, or about 45 cm;about 45 cm to about 75 cm, about 70 cm, about 65 cm, about 60 cm, about55 cm, or about 50 cm; about 50 cm to about 75 cm, about 70 cm, about 65cm, about 60 cm, or about 55 cm; about 55 cm to about 75 cm, about 70cm, about 65 cm, or about 60 cm; about 60 cm to about 75 cm, about 70cm, or about 65 cm; about 65 cm to about 75 cm or about 70 cm; or about70 cm to about 75 cm.

Additional examples of conduits and properties of conduits that can beused in the second circuit system are well-known by those in the art.

In some examples, the second circuit system includes one, two, three,four, or five ports. In some examples, the at least one port in thesecond circuit system can be a valve. In other examples, the at leastone port in the second circuit system can be an injection port or canhave a ribbed threading. The at least one port in the second circuitsystem can be any type of port commonly known in the art.

In some examples, the second circuit system can further include a pump(e.g., a low-turbulence pump, e.g., a peristaltic pump) disposed in theconnecting conduit and/or the conduit in second system. In someembodiments, the second circuit system does not include a holding tank.In some embodiments, the connecting conduit is not fluidly connected toa holding tank.

In some examples, step (f) includes continuously flowing (e.g.,unidirectionally or bidirectionally flowing) the collected fluid of step(d) into the second system (using the connecting conduit) at a rate ofabout 0.05 mL/minute to about 100 L/minute, about 90 L/minute, about 80L/minute, about 70 L/minute, about 60 L/minute, about 50 L/minute, about40 L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute,about 5 L/minute, about 1 L/minute, about 950 mL/minute, about 900mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, about 7.0mL/minute, about 6.5 mL/minute, about 6.0 mL/minute, about 5.5mL/minute, about 5.0 mL/minute, about 4.5 mL/minute, about 4.0mL/minute, about 3.5 mL/minute, about 3.0 mL/minute, about 2.5mL/minute, about 2.0 mL/minute, about 1.5 mL/minute, about 1.0mL/minute, about 0.5 mL/minute, or about 0.1 mL/minute; about 0.1mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, about 6.5mL/minute, about 6.0 mL/minute, about 5.5 mL/minute, about 5.0mL/minute, about 4.5 mL/minute, about 4.0 mL/minute, about 3.5mL/minute, about 3.0 mL/minute, about 2.5 mL/minute, about 2.0mL/minute, about 1.5 mL/minute, about 1.0 mL/minute, or about 0.5mL/minute; about 0.5 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, about 7.0mL/minute, about 6.5 mL/minute, about 6.0 mL/minute, about 5.5mL/minute, about 5.0 mL/minute, about 4.5 mL/minute, about 4.0mL/minute, about 3.5 mL/minute, about 3.0 mL/minute, about 2.5mL/minute, about 2.0 mL/minute, about 1.5 mL/minute, or about 1.0mL/minute; about 1.0 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, about 7.0mL/minute, about 6.5 mL/minute, about 6.0 mL/minute, about 5.5mL/minute, about 5.0 mL/minute, about 4.5 mL/minute, about 4.0mL/minute, about 3.5 mL/minute, about 3.0 mL/minute, about 2.5mL/minute, about 2.0 mL/minute, or about 1.5 mL/minute; about 1.5mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, about 6.5mL/minute, about 6.0 mL/minute, about 5.5 mL/minute, about 5.0mL/minute, about 4.5 mL/minute, about 4.0 mL/minute, about 3.5mL/minute, about 3.0 mL/minute, about 2.5 mL/minute, or about 2.0mL/minute; about 2.0 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, about 7.0mL/minute, about 6.5 mL/minute, about 6.0 mL/minute, about 5.5mL/minute, about 5.0 mL/minute, about 4.5 mL/minute, about 4.0mL/minute, about 3.5 mL/minute, about 3.0 mL/minute, or about 2.5mL/minute; about 2.5 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, about 7.0mL/minute, about 6.5 mL/minute, about 6.0 mL/minute, about 5.5mL/minute, about 5.0 mL/minute, about 4.5 mL/minute, about 4.0mL/minute, about 3.5 mL/minute, or about 3.0 mL/minute; about 3.0mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, about 6.5mL/minute, about 6.0 mL/minute, about 5.5 mL/minute, about 5.0mL/minute, about 4.5 mL/minute, about 4.0 mL/minute, or about 3.5mL/minute; about 3.5 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, about 7.0mL/minute, about 6.5 mL/minute, about 6.0 mL/minute, about 5.5mL/minute, about 5.0 mL/minute, about 4.5 mL/minute, or about 4.0mL/minute; about 4.0 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, about 7.0mL/minute, about 6.5 mL/minute, about 6.0 mL/minute, about 5.5mL/minute, about 5.0 mL/minute, or about 4.5 mL/minute; about 4.5mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, about 6.5mL/minute, about 6.0 mL/minute, about 5.5 mL/minute, or about 5.0mL/minute; about 5.0 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, about 7.0mL/minute, about 6.5 mL/minute, about 6.0 mL/minute, or about 5.5mL/minute; about 5.5 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, about 7.0mL/minute, about 6.5 mL/minute, or about 6.0 mL/minute; about 6.0mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, about 8.0mL/minute, about 7.5 mL/minute, about 7.0 mL/minute, or about 6.5mL/minute; about 6.5 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, about 7.5 mL/minute, or about 7.0mL/minute; about 7.0 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,about 10 mL/minute, about 9.5 mL/minute, about 9.0 mL/minute, about 8.5mL/minute, about 8.0 mL/minute, or about 7.5 mL/minute; about 7.5mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 24 mL/minute,about 22 mL/minute, about 20 mL/minute, about 18 mL/minute, about 16mL/minute, about 14 mL/minute, about 12 mL/minute, about 10 mL/minute,about 9.5 mL/minute, about 9.0 mL/minute, about 8.5 mL/minute, or about8.0 mL/minute; about 8.0 mL/minute to about 100 L/minute, about 90L/minute, about 80 L/minute, about 70 L/minute, about 60 L/minute, about50 L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute,about 10 L/minute, about 5 L/minute, about 1 L/minute, about 950mL/minute, about 900 mL/minute, about 850 mL/minute, about 800mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute, about 24 mL/minute, about 22 mL/minute, about 20mL/minute, about 18 mL/minute, about 16 mL/minute, about 14 mL/minute,about 12 mL/minute, about 10 mL/minute, about 9.5 mL/minute, about 9.0mL/minute, or about 8.5 mL/minute; about 8.5 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,about 800 mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute, about 24 mL/minute, about 22 mL/minute, about 20mL/minute, about 18 mL/minute, about 16 mL/minute, about 14 mL/minute,about 12 mL/minute, about 10 mL/minute, about 9.5 mL/minute, or about9.0 mL/minute; about 9.0 mL/minute to about 100 L/minute, about 90L/minute, about 80 L/minute, about 70 L/minute, about 60 L/minute, about50 L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute,about 10 L/minute, about 5 L/minute, about 1 L/minute, about 950mL/minute, about 900 mL/minute, about 850 mL/minute, about 800mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute, about 24 mL/minute, about 22 mL/minute, about 20mL/minute, about 18 mL/minute, about 16 mL/minute, about 14 mL/minute,about 12 mL/minute, about 10 mL/minute, or about 9.5 mL/minute; about9.5 mL/minute to about 100 L/minute, about 90 L/minute, about 80L/minute, about 70 L/minute, about 60 L/minute, about 50 L/minute, about40 L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute,about 5 L/minute, about 1 L/minute, about 950 mL/minute, about 900mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, about 16 mL/minute, about 14 mL/minute, about 12 mL/minute,or about 10 mL/minute; about 10 mL/minute to about 100 L/minute, about90 L/minute, about 80 L/minute, about 70 L/minute, about 60 L/minute,about 50 L/minute, about 40 L/minute, about 30 L/minute, about 20L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about950 mL/minute, about 900 mL/minute, about 850 mL/minute, about 800mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute, about 24 mL/minute, about 22 mL/minute, about 20mL/minute, about 18 mL/minute, about 16 mL/minute, about 14 mL/minute,or about 12 mL/minute; about 12 mL/minute to about 100 L/minute, about90 L/minute, about 80 L/minute, about 70 L/minute, about 60 L/minute,about 50 L/minute, about 40 L/minute, about 30 L/minute, about 20L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about950 mL/minute, about 900 mL/minute, about 850 mL/minute, about 800mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute, about 24 mL/minute, about 22 mL/minute, about 20mL/minute, about 18 mL/minute, about 16 mL/minute, or about 14mL/minute; about 14 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, about 22 mL/minute, about 20 mL/minute, about 18mL/minute, or about 16 mL/minute; about 16 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,about 800 mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute, about 24 mL/minute, about 22 mL/minute, about 20mL/minute, or about 18 mL/minute; about 18 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,about 800 mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute, about 24 mL/minute, about 22 mL/minute, or about 20mL/minute; about 20 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 24 mL/minute, or about 22 mL/minute; about 22 mL/minute to about100 L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute,about 60 L/minute, about 50 L/minute, about 40 L/minute, about 30L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute or about 24 mL/minute;about 23 mL/minute to about 100 L/minute, about 90 L/minute, about 80L/minute, about 70 L/minute, about 60 L/minute, about 50 L/minute, about40 L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute,about 5 L/minute, about 1 L/minute, about 950 mL/minute, about 900mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 50 mL/minute, or about 25mL/minute; about 25 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, or about 50 mL/minute; about 50mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, or about 100mL/minute; about 100 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, or about 150mL/minute; about 150 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, or about 200 mL/minute; about 200mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, or about 250mL/minute; about 250 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, or about 300mL/minute; about 300 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, or about 350 mL/minute; about 350mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, or about 400mL/minute; about 400 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, or about 450mL/minute; about 450 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, or about 500 mL/minute; about 500mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, or about 550mL/minute; about 550 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, or about 600mL/minute; about 600 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, or about 650 mL/minute; about 650mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, or about700 mL/minute; about 700 mL/minute to about 100 L/minute, about 90L/minute, about 80 L/minute, about 70 L/minute, about 60 L/minute, about50 L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute,about 10 L/minute, about 5 L/minute, about 1 L/minute, about 950mL/minute, about 900 mL/minute, about 850 mL/minute, about 800mL/minute, or about 750 mL/minute; about 750 mL/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 950 mL/minute, about 900 mL/minute, about 850 mL/minute,or about 800 mL/minute; about 800 mL/minute to about 100 L/minute, about90 L/minute, about 80 L/minute, about 70 L/minute, about 60 L/minute,about 50 L/minute, about 40 L/minute, about 30 L/minute, about 20L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about950 mL/minute, about 900 mL/minute, or about 850 mL/minute; about 850mL/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, or about 900mL/minute; about 900 mL/minute to about 100 L/minute, about 90 L/minute,about 80 L/minute, about 70 L/minute, about 60 L/minute, about 50L/minute, about 40 L/minute, about 30 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, or about 950 mL/minute;about 950 mL/minute to about 100 L/minute, about 90 L/minute, about 80L/minute, about 70 L/minute, about 60 L/minute, about 50 L/minute, about40 L/minute, about 30 L/minute, about 20 L/minute, about 10 L/minute,about 5 L/minute, or about 1 L/minute; about 1 L/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, about 50 L/minute, about 40 L/minute, about 30 L/minute,about 20 L/minute, about 10 L/minute, or about 5 L/minute; about 5L/minute to about 100 L/minute, about 90 L/minute, about 80 L/minute,about 70 L/minute, about 60 L/minute, about 50 L/minute, about 40L/minute, about 30 L/minute, about 20 L/minute, or about 10 L/minute;about 10 L/minute to about 100 L/minute, about 90 L/minute, about 80L/minute, about 70 L/minute, about 60 L/minute, about 50 L/minute, about40 L/minute, about 30 L/minute, or about 20 L/minute; about 20 L/minuteto about 100 L/minute, about 90 L/minute, about 80 L/minute, about 70L/minute, about 60 L/minute, about 50 L/minute, about 40 L/minute, orabout 30 L/minute; about 30 L/minute to about 100 L/minute, about 90L/minute, about 80 L/minute, about 70 L/minute, about 60 L/minute, about50 L/minute, or about 40 L/minute; about 40 L/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, about60 L/minute, or about 50 L/minute; about 50 L/minute to about 100L/minute, about 90 L/minute, about 80 L/minute, about 70 L/minute, orabout 60 L/minute; about 60 L/minute to about 100 L/minute, about 90L/minute, about 80 L/minute, or about 70 L/minute; about 70 L/minute toabout 100 L/minute, about 90 L/minute, or about 80 L/minute; about 80L/minute to about 100 L/minute or about 90 L/minute; or about 90L/minute to about 100 L/minute.

In some examples, the filtrate collected from the TFVF unit in step (f)includes a concentration of recombinant therapeutic protein of about 0.1mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL,about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL,about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL,about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL,about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL,about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14 mg/mL, about 12mg/mL, about 10 mg/mL, about 9 mg/mL, about 8 mg/mL, about 7 mg/mL,about 6 mg/mL, about 5 mg/mL, about 4 mg/mL, about 3 mg/mL, about 2.5mg/mL, about 2.0 mg/mL, about 1.5 mg/mL, about 1.0 mg/mL, or about 0.5mg/mL; about 0.5 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL,about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL,about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL,about 22 mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14mg/mL, about 12 mg/mL, about 10 mg/mL, about 9 mg/mL, about 8 mg/mL,about 7 mg/mL, about 6 mg/mL, about 5 mg/mL, about 4 mg/mL, about 3mg/mL, about 2.5 mg/mL, about 2.0 mg/mL, about 1.5 mg/mL, or about 1.0mg/mL; about 1.0 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL,about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL,about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL,about 22 mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14mg/mL, about 12 mg/mL, about 10 mg/mL, about 9 mg/mL, about 8 mg/mL,about 7 mg/mL, about 6 mg/mL, about 5 mg/mL, about 4 mg/mL, about 3mg/mL, about 2.5 mg/mL, about 2.0 mg/mL, or about 1.5 mg/mL; about 1.5mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL,about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL,about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL,about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL,about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL,about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14 mg/mL, about 12mg/mL, about 10 mg/mL, about 9 mg/mL, about 8 mg/mL, about 7 mg/mL,about 6 mg/mL, about 5 mg/mL, about 4 mg/mL, about 3 mg/mL, about 2.5mg/mL, or about 2.0 mg/mL; about 2.0 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72 mg/mL,about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL, about 62mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54 mg/mL,about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL,about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL, about 26mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL, about 18 mg/mL,about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, about 10 mg/mL, about 9mg/mL, about 8 mg/mL, about 7 mg/mL, about 6 mg/mL, about 5 mg/mL, about4 mg/mL, about 3 mg/mL, or about 2.5 mg/mL; about 2.5 mg/mL to about 100mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL,about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL,about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL,about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL,about 18 mg/mL, about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, about 10mg/mL, about 9 mg/mL, about 8 mg/mL, about 7 mg/mL, about 6 mg/mL, about5 mg/mL, about 4 mg/mL, or about 3 mg/mL; about 3 mg/mL to about 100mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL,about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL,about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL,about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL,about 18 mg/mL, about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, about 10mg/mL, about 9 mg/mL, about 8 mg/mL, about 7 mg/mL, about 6 mg/mL, about5 mg/mL, or about 4 mg/mL; about 4 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72 mg/mL,about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL, about 62mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54 mg/mL,about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL,about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL, about 26mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL, about 18 mg/mL,about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, about 10 mg/mL, about 9mg/mL, about 8 mg/mL, about 7 mg/mL, about 6 mg/mL, or about 5 mg/mL;about 5 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL,about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL,about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL, about 58mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50 mg/mL,about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL,about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14 mg/mL,about 12 mg/mL, about 10 mg/mL, about 9 mg/mL, about 8 mg/mL, about 7mg/mL, or about 6 mg/mL; about 6 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72 mg/mL,about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL, about 62mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54 mg/mL,about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL,about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL, about 26mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL, about 18 mg/mL,about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, about 10 mg/mL, about 9mg/mL, about 8 mg/mL, or about 7 mg/mL; about 7 mg/mL to about 100mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL,about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL,about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL,about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL,about 18 mg/mL, about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, about 10mg/mL, about 9 mg/mL, or about 8 mg/mL; about 8 mg/mL to about 100mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL,about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL,about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL,about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL,about 18 mg/mL, about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, about 10mg/mL, or about 9 mg/mL; about 9 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72 mg/mL,about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL, about 62mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54 mg/mL,about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL,about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL, about 26mg/mL, about 24 mg/mL, about 22 mg/mL, about 20 mg/mL, about 18 mg/mL,about 16 mg/mL, about 14 mg/mL, about 12 mg/mL, or about 10 mg/mL; about10 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL,about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL,about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL,about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL,about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL,about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, about 14 mg/mL, or about12 mg/mL; about 12 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL,about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL,about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL,about 22 mg/mL, about 20 mg/mL, about 18 mg/mL, about 16 mg/mL, or about14 mg/mL; about 14 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL,about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL,about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL,about 22 mg/mL, about 20 mg/mL, about 18 mg/mL, or about 16 mg/mL; about16 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL,about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL,about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL,about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL,about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, about 22 mg/mL,about 20 mg/mL, or about 18 mg/mL; about 18 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL,about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL,about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL, about 28 mg/mL,about 26 mg/mL, about 24 mg/mL, about 22 mg/mL, or about 20 mg/mL; about20 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL,about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL,about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL,about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL,about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30mg/mL, about 28 mg/mL, about 26 mg/mL, about 24 mg/mL, or about 22mg/mL; about 22 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL,about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL,about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32mg/mL, about 30 mg/mL, about 28 mg/mL, about 26 mg/mL, or about 24mg/mL; about 24 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL,about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL,about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL, about 32mg/mL, about 30 mg/mL, about 28 mg/mL, or about 26 mg/mL; about 26 mg/mLto about 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL,about 64 mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56mg/mL, about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL,about 46 mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38mg/mL, about 36 mg/mL, about 34 mg/mL, about 32 mg/mL, about 30 mg/mL,or about 28 mg/mL; about 28 mg/mL to about 100 mg/mL, about 98 mg/mL, 96mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL,about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78mg/mL, about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL,about 68 mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60mg/mL, about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL,about 50 mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42mg/mL, about 40 mg/mL, about 38 mg/mL, about 36 mg/mL, about 34 mg/mL,about 32 mg/mL, or about 30 mg/mL; about 30 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL,about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL,about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, about 36mg/mL, about 34 mg/mL, or about 32 mg/mL; about 32 mg/mL to about 100mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL,about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL,about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46mg/mL, about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL,about 36 mg/mL, or about 34 mg/mL; about 34 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL,about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL,about 44 mg/mL, about 42 mg/mL, about 40 mg/mL, about 38 mg/mL, or about36 mg/mL; about 36 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL,about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50mg/mL, about 48 mg/mL, about 46 mg/mL, about 44 mg/mL, about 42 mg/mL,about 40 mg/mL, or about 38 mg/mL; about 38 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL,about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, about 46 mg/mL,about 44 mg/mL, about 42 mg/mL, or about 40 mg/mL; about 40 mg/mL toabout 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL,about 64 mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56mg/mL, about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL,about 46 mg/mL, about 44 mg/mL, or about 42 mg/mL; about 42 mg/mL toabout 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL,about 64 mg/mL, about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56mg/mL, about 54 mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL,about 46 mg/mL, or about 44 mg/mL; about 44 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL,about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54mg/mL, about 52 mg/mL, about 50 mg/mL, about 48 mg/mL, or about 46mg/mL; about 46 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL,about 58 mg/mL, about 56 mg/mL, about 54 mg/mL, about 52 mg/mL, about 50mg/mL, or about 48 mg/mL; about 48 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72 mg/mL,about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL, about 62mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54 mg/mL,about 52 mg/mL, or about 50 mg/mL; about 50 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL,about 62 mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, about 54mg/mL, or about 52 mg/mL; about 52 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72 mg/mL,about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL, about 62mg/mL, about 60 mg/mL, about 58 mg/mL, about 56 mg/mL, or about 54mg/mL; about 54 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, about 66 mg/mL, about 64 mg/mL, about 62 mg/mL, about 60 mg/mL,about 58 mg/mL, or about 56 mg/mL; about 56 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, about 64 mg/mL,about 62 mg/mL, about 60 mg/mL, or about 58 mg/mL; about 58 mg/mL toabout 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL,about 64 mg/mL, about 62 mg/mL, or about 60 mg/mL; about 60 mg/mL toabout 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74mg/mL, about 72 mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL,about 64 mg/mL, or about 62 mg/mL; about 62 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, about 70 mg/mL, about 68 mg/mL, about 66 mg/mL, or about 64mg/mL; about 64 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, about 74 mg/mL, about 72 mg/mL, about 70 mg/mL, about 68mg/mL, or about 66 mg/mL; about 66 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72 mg/mL,about 70 mg/mL, or about 68 mg/mL; about 68 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, about 72mg/mL, or about 70 mg/mL; about 70 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL, about 80mg/mL, about 78 mg/mL, about 76 mg/mL, about 74 mg/mL, or about 72mg/mL; about 72 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, about 84 mg/mL, about 82 mg/mL, about 80 mg/mL, about 78 mg/mL,about 76 mg/mL, or about 74 mg/mL; about 74 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, about 82 mg/mL,about 80 mg/mL, about 78 mg/mL, or about 76 mg/mL; about 76 mg/mL toabout 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, about 80 mg/mL, or about 78 mg/mL; about 78 mg/mL toabout 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92mg/mL, about 90 mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL,about 82 mg/mL, or about 80 mg/mL; about 80 mg/mL to about 100 mg/mL,about 98 mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90mg/mL, about 88 mg/mL, about 86 mg/mL, about 84 mg/mL, or about 82mg/mL; about 82 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about 88 mg/mL, about 86mg/mL, or about 84 mg/mL; about 84 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, about88 mg/mL, or about 86 mg/mL; about 86 mg/mL to about 100 mg/mL, about 98mg/mL, 96 mg/mL, about 94 mg/mL, about 92 mg/mL, about 90 mg/mL, orabout 88 mg/mL; about 88 mg/mL to about 100 mg/mL, about 98 mg/mL, 96mg/mL, about 94 mg/mL, about 92 mg/mL, or about 90 mg/mL; about 90 mg/mLto about 100 mg/mL, about 98 mg/mL, 96 mg/mL, about 94 mg/mL, or about92 mg/mL; about 92 mg/mL to about 100 mg/mL, about 98 mg/mL, 96 mg/mL,or about 94 mg/mL; about 94 mg/mL to about 100 mg/mL, about 98 mg/mL, or96 mg/mL; about 96 mg/mL to about 100 mg/mL or about 98 mg/mL; or about98 mg/mL to about 100 mg/mL.

In some examples of these methods, the third period of time can be about0.1 minute to about 12 hours, about 11.5 hours, about 11 hours, about10.5 hours, about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5hours, about 8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours,about 6.0 hours, about 5.5 hours, about 5.0 hours, about 4.5 hours,about 4.0 hours, about 3.5 hours, about 3.0 hours, about 2.5 hours,about 2.0 hours, about 1.5 hours, about 1.0 hour, about 55 minutes,about 50 minutes, about 45 minutes, about 40 minutes, about 35 minutes,about 30 minutes, about 25 minutes, about 20 minutes, about 15 minutes,about 10 minutes, about 5 minutes, about 2 minutes, or about 1 minute;about 1 minute to about 12 hours, about 11.5 hours, about 11 hours,about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0 hours,about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours, about6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours, about 4.5hours, about 4.0 hours, about 3.5 hours, about 3.0 hours, about 2.5hours, about 2.0 hours, about 1.5 hours, about 1.0 hour, about 55minutes, about 50 minutes, about 45 minutes, about 40 minutes, about 35minutes, about 30 minutes, about 25 minutes, about 20 minutes, about 15minutes, about 10 minutes, about 5 minutes, or about 2 minutes; about 2minutes to about 12 hours, about 11.5 hours, about 11 hours, about 10.5hours, about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5hours, about 8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours,about 6.0 hours, about 5.5 hours, about 5.0 hours, about 4.5 hours,about 4.0 hours, about 3.5 hours, about 3.0 hours, about 2.5 hours,about 2.0 hours, about 1.5 hours, about 1.0 hour, about 55 minutes,about 50 minutes, about 45 minutes, about 40 minutes, about 35 minutes,about 30 minutes, about 25 minutes, about 20 minutes, about 15 minutes,about 10 minutes, or about 5 minutes; about 5 minutes to about 12 hours,about 11.5 hours, about 11 hours, about 10.5 hours, about 10 hours,about 9.5 hours, about 9.0 hours, about 8.5 hours, about 8.0 hours,about 7.5 hours, about 7 hours, about 6.5 hours, about 6.0 hours, about5.5 hours, about 5.0 hours, about 4.5 hours, about 4.0 hours, about 3.5hours, about 3.0 hours, about 2.5 hours, about 2.0 hours, about 1.5hours, about 1.0 hour, about 55 minutes, about 50 minutes, about 45minutes, about 40 minutes, about 35 minutes, about 30 minutes, about 25minutes, about 20 minutes, about 15 minutes, or about 10 minutes; about10 minutes to about 12 hours, about 11.5 hours, about 11 hours, about10.5 hours, about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5hours, about 8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours,about 6.0 hours, about 5.5 hours, about 5.0 hours, about 4.5 hours,about 4.0 hours, about 3.5 hours, about 3.0 hours, about 2.5 hours,about 2.0 hours, about 1.5 hours, about 1.0 hour, about 55 minutes,about 50 minutes, about 45 minutes, about 40 minutes, about 35 minutes,about 30 minutes, about 25 minutes, about 20 minutes, or about 15minutes; about 15 minutes to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours,about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.0 hour, about55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, about35 minutes, about 30 minutes, about 25 minutes, or about 20 minutes;about 20 minutes to about 12 hours, about 11.5 hours, about 11 hours,about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0 hours,about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours, about6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours, about 4.5hours, about 4.0 hours, about 3.5 hours, about 3.0 hours, about 2.5hours, about 2.0 hours, about 1.5 hours, about 1.0 hour, about 55minutes, about 50 minutes, about 45 minutes, about 40 minutes, about 35minutes, about 30 minutes, or about 25 minutes; about 25 minutes toabout 12 hours, about 11.5 hours, about 11 hours, about 10.5 hours,about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5 hours, about8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours, about 6.0hours, about 5.5 hours, about 5.0 hours, about 4.5 hours, about 4.0hours, about 3.5 hours, about 3.0 hours, about 2.5 hours, about 2.0hours, about 1.5 hours, about 1.0 hour, about 55 minutes, about 50minutes, about 45 minutes, about 40 minutes, about 35 minutes, or about30 minutes; about 30 minutes to about 12 hours, about 11.5 hours, about11 hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours,about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.0 hour, about55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, orabout 35 minutes; about 35 minutes to about 12 hours, about 11.5 hours,about 11 hours, about 10.5 hours, about 10 hours, about 9.5 hours, about9.0 hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7hours, about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0hours, about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0hours, about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.0hour, about 55 minutes, about 50 minutes, about 45 minutes, or about 40minutes; about 40 minutes to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours,about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.0 hour, about55 minutes, about 50 minutes, or about 45 minutes; about 45 minutes toabout 12 hours, about 11.5 hours, about 11 hours, about 10.5 hours,about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5 hours, about8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours, about 6.0hours, about 5.5 hours, about 5.0 hours, about 4.5 hours, about 4.0hours, about 3.5 hours, about 3.0 hours, about 2.5 hours, about 2.0hours, about 1.5 hours, about 1.0 hour, about 55 minutes, or about 50minutes; about 50 minutes to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours,about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.0 hour, orabout 55 minutes; about 55 minutes to about 12 hours, about 11.5 hours,about 11 hours, about 10.5 hours, about 10 hours, about 9.5 hours, about9.0 hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7hours, about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0hours, about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0hours, about 2.5 hours, about 2.0 hours, about 1.5 hours, or about 1.0hour; about 1.0 hour to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours,about 2.5 hours, about 2.0 hours, or about 1.5 hours; about 1.5 hours toabout 12 hours, about 11.5 hours, about 11 hours, about 10.5 hours,about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5 hours, about8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours, about 6.0hours, about 5.5 hours, about 5.0 hours, about 4.5 hours, about 4.0hours, about 3.5 hours, about 3.0 hours, about 2.5 hours, or about 2.0hours; about 2.0 hours to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, about 3.5 hours, about 3.0 hours, orabout 2.5 hours; about 2.5 hours to about 12 hours, about 11.5 hours,about 11 hours, about 10.5 hours, about 10 hours, about 9.5 hours, about9.0 hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7hours, about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0hours, about 4.5 hours, about 4.0 hours, about 3.5 hours, or about 3.0hours; about 3.0 hours to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours,about 4.5 hours, about 4.0 hours, or about 3.5 hours; about 3.5 hours toabout 12 hours, about 11.5 hours, about 11 hours, about 10.5 hours,about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5 hours, about8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours, about 6.0hours, about 5.5 hours, about 5.0 hours, about 4.5 hours, or about 4.0hours; about 4.0 hours to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, about 5.5 hours, about 5.0 hours, orabout 4.5 hours; about 4.5 hours to about 12 hours, about 11.5 hours,about 11 hours, about 10.5 hours, about 10 hours, about 9.5 hours, about9.0 hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7hours, about 6.5 hours, about 6.0 hours, about 5.5 hours, or about 5.0hours; about 5.0 hours to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,about 6.5 hours, about 6.0 hours, or about 5.5 hours; about 5.5 hours toabout 12 hours, about 11.5 hours, about 11 hours, about 10.5 hours,about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5 hours, about8.0 hours, about 7.5 hours, about 7 hours, about 6.5 hours, or about 6.0hours; about 6.0 hours to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, about 7 hours,or about 6.5 hours; about 6.5 hours to about 12 hours, about 11.5 hours,about 11 hours, about 10.5 hours, about 10 hours, about 9.5 hours, about9.0 hours, about 8.5 hours, about 8.0 hours, about 7.5 hours, or about 7hours; about 7 hours to about 12 hours, about 11.5 hours, about 11hours, about 10.5 hours, about 10 hours, about 9.5 hours, about 9.0hours, about 8.5 hours, about 8.0 hours, or about 7.5 hours; about 7.5hours to about 12 hours, about 11.5 hours, about 11 hours, about 10.5hours, about 10 hours, about 9.5 hours, about 9.0 hours, about 8.5hours, or about 8.0 hours; about 8.0 hours to about 12 hours, about 11.5hours, about 11 hours, about 10.5 hours, about 10 hours, about 9.5hours, about 9.0 hours, or about 8.5 hours; about 8.5 hours to about 12hours, about 11.5 hours, about 11 hours, about 10.5 hours, about 10hours, about 9.5 hours, or about 9.0 hours; about 9.0 hours to about 12hours, about 11.5 hours, about 11 hours, about 10.5 hours, about 10hours, or about 9.5 hours; about 9.5 hours to about 12 hours, about 11.5hours, about 11 hours, about 10.5 hours, or about 10 hours; about 10hours to about 12 hours, about 11.5 hours, about 11 hours, or about 10.5hours; about 10.5 hours to about 12 hours, about 11.5 hours, or about 11hours; about 11 hours to about 12 hours, or about 11.5 hours.

In some examples of these methods, the third period of time can be,e.g., about 12 hours to about 72 hours, about 12 hours to about 69hours, about 12 hours to about 66 hours, about 12 hours to about 63hours, about 12 hours to about 60 hours, about 12 hours to about 57hours, about 12 hours to about 54 hours, about 12 hours to about 51hours, about 12 hours to about 48 hours, about 12 hours to about 45hours, about 12 hours to about 42 hours, about 12 hours to about 39hours, about 12 hours to about 36 hours, about 12 hours to about 33hours, about 12 hours to about 30 hours, about 12 hours to about 27hours, about 12 hours to about 24 hours, about 12 hours to about 21hours, about 12 hours to about 18 hours, about 12 hours to about 15hours, about 15 hours to about 72 hours, about 15 hours to about 69hours, about 15 hours to about 66 hours, about 15 hours to about 63hours, about 15 hours to about 60 hours, about 15 hours to about 57hours, about 15 hours to about 54 hours, about 15 hours to about 51hours, about 15 hours to about 48 hours, about 15 hours to about 45hours, about 15 hours to about 42 hours, about 15 hours to about 39hours, about 15 hours to about 36 hours, about 15 hours to about 33hours, about 15 hours to about 30 hours, about 15 hours to about 27hours, about 15 hours to about 24 hours, about 15 hours to about 21hours, about 15 hours to about 18 hours, about 18 hours to about 72hours, about 18 hours to about 69 hours, about 18 hours to about 66hours, about 18 hours to about 63 hours, about 18 hours to about 60hours, about 18 hours to about 57 hours, about 18 hours to about 54hours, about 18 hours to about 51 hours, about 18 hours to about 48hours, about 18 hours to about 45 hours, about 18 hours to about 42hours, about 18 hours to about 39 hours, about 18 hours to about 36hours, about 18 hours to about 33 hours, about 18 hours to about 30hours, about 18 hours to about 27 hours, about 18 hours to about 24hours, about 18 hours to about 21 hours, about 21 hours to about 72hours, about 21 hours to about 69 hours, about 21 hours to about 66hours, about 21 hours to about 63 hours, about 21 hours to about 60hours, about 21 hours to about 57 hours, about 21 hours to about 54hours, about 21 hours to about 51 hours, about 21 hours to about 48hours, about 21 hours to about 45 hours, about 21 hours to about 42hours, about 21 hours to about 39 hours, about 21 hours to about 36hours, about 21 hours to about 33 hours, about 21 hours to about 30hours, about 21 hours to about 27 hours, about 21 hours to about 24hours, about 24 hours to about 72 hours, about 24 hours to about 69hours, about 24 hours to about 66 hours, about 24 hours to about 63hours, about 24 hours to about 60 hours, about 24 hours to about 57hours, about 24 hours to about 54 hours, about 24 hours to about 51hours, about 24 hours to about 48 hours, about 24 hours to about 45hours, about 24 hours to about 42 hours, about 24 hours to about 39hours, about 24 hours to about 36 hours, about 24 hours to about 33hours, about 24 hours to about 30 hours, about 24 hours to about 27hours, about 27 hours to about 72 hours, about 27 hours to about 69hours, about 27 hours to about 66 hours, about 27 hours to about 63hours, about 27 hours to about 60 hours, about 27 hours to about 57hours, about 27 hours to about 54 hours, about 27 hours to about 51hours, about 27 hours to about 48 hours, about 27 hours to about 45hours, about 27 hours to about 42 hours, about 27 hours to about 39hours, about 27 hours to about 36 hours, about 27 hours to about 33hours, about 27 hours to about 30 hours, about 30 hours to about 72hours, about 30 hours to about 69 hours, about 30 hours to about 66hours, about 30 hours to about 63 hours, about 30 hours to about 60hours, about 30 hours to about 57 hours, about 30 hours to about 54hours, about 30 hours to about 51 hours, about 30 hours to about 48hours, about 30 hours to about 45 hours, about 30 hours to about 42hours, about 30 hours to about 39 hours, about 30 hours to about 36hours, about 30 hours to about 33 hours, about 33 hours to about 72hours, about 33 hours to about 69 hours, about 33 hours to about 66hours, about 33 hours to about 63 hours, about 33 hours to about 60hours, about 33 hours to about 57 hours, about 33 hours to about 54hours, about 33 hours to about 51 hours, about 33 hours to about 48hours, about 33 hours to about 45 hours, about 33 hours to about 42hours, about 33 hours to about 39 hours, about 33 hours to about 36hours, about 36 hours to about 72 hours, about 36 hours to about 69hours, about 36 hours to about 66 hours, about 36 hours to about 63hours, about 36 hours to about 60 hours, about 36 hours to about 57hours, about 36 hours to about 54 hours, about 36 hours to about 51hours, about 36 hours to about 48 hours, about 36 hours to about 45hours, about 36 hours to about 42 hours, about 36 hours to about 39hours, about 39 hours to about 72 hours, about 39 hours to about 69hours, about 39 hours to about 66 hours, about 39 hours to about 63hours, about 39 hours to about 60 hours, about 39 hours to about 57hours, about 39 hours to about 54 hours, about 39 hours to about 51hours, about 39 hours to about 48 hours, about 39 hours to about 45hours, about 39 hours to about 42 hours, about 42 hours to about 72hours, about 42 hours to about 69 hours, about 42 hours to about 66hours, about 42 hours to about 63 hours, about 42 hours to about 60hours, about 42 hours to about 57 hours, about 42 hours to about 54hours, about 42 hours to about 51 hours, about 42 hours to about 48hours, about 42 hours to about 45 hours, about 45 hours to about 72hours, about 45 hours to about 69 hours, about 45 hours to about 66hours, about 45 hours to about 63 hours, about 45 hours to about 60hours, about 45 hours to about 57 hours, about 45 hours to about 54hours, about 45 hours to about 51 hours, about 45 hours to about 48hours, about 48 hours to about 72 hours, about 48 hours to about 69hours, about 48 hours to about 66 hours, about 48 hours to about 63hours, about 48 hours to about 60 hours, about 48 hours to about 57hours, about 48 hours to about 54 hours, about 48 hours to about 51hours, about 51 hours to about 72 hours, about 51 hours to about 69hours, about 51 hours to about 66 hours, about 51 hours to about 63hours, about 51 hours to about 60 hours, about 51 hours to about 57hours, about 51 hours to about 54 hours, about 54 hours to about 72hours, about 54 hours to about 69 hours, about 54 hours to about 66hours, about 54 hours to about 63 hours, about 54 hours to about 60hours, about 54 hours to about 57 hours, about 57 hours to about 72hours, about 57 hours to about 69 hours, about 57 hours to about 66hours, about 57 hours to about 63 hours, about 57 hours to about 60hours, about 60 hours to about 72 hours, about 60 hours to about 69hours, about 60 hours to about 66 hours, about 60 hours to about 63hours, about 63 hours to about 72 hours, about 63 hours to about 69hours, about 63 hours to about 66 hours, about 66 hours to about 72hours, about 66 hours to about 69 hours, or about 69 hours to about 72hours.

In some embodiments of these methods, the collected fluid of step (d)that is continuously flowed into the second circuit system including theTFVF unit can include a total virus titer of about 10⁻¹ to about 10⁻²⁰,about 10⁻¹ to about 10⁻¹⁹, about 10⁻¹ to about 10⁻¹⁸, about 10⁻¹ toabout 10⁻¹⁷, about 10⁻¹ to about 10⁻¹⁶, about 10⁻¹ to about 10⁻¹⁵, about10⁻¹ to about 10⁻¹⁴, about 10⁻¹ to about 10⁻¹³, about 10⁻¹ to about10⁻¹², about 10⁻¹ to about 10⁻¹¹, about 10⁻¹ to about 10⁻¹⁰, about 10⁻¹to about 10⁻⁹, about 10⁻¹ to about 10⁻⁸, about 10⁻¹ to about 10⁻⁷, about10⁻¹ to about 10⁻⁶, about 10⁻¹ to about 10⁻⁵, about 10⁻¹ to about 10⁻⁴,about 10⁻¹ to about 10⁻³, about 10⁻¹ to about 10⁻², about 10⁻² to about10⁻²⁰, about 10⁻² to about 10⁻¹⁹, about 10⁻² to about 10⁻¹⁸, about 10⁻²to about 10⁻¹⁷, about 10⁻² to about 10⁻¹⁶, about 10⁻² to about 10⁻¹⁵,about 10⁻² to about 10⁻¹⁴, about 10⁻² to about 10⁻¹³, about 10⁻² toabout 10⁻¹², about 10⁻² to about 10⁻¹¹, about 10⁻² to about 10⁻¹⁰, about10⁻² to about 10⁻⁹, about 10⁻² to about 10⁻⁸, about 10⁻² to about 10⁻⁷,about 10⁻² to about 10⁻⁶, about 10⁻² to about 10⁻⁵, about 10⁻² to about10⁻⁴, about 10⁻² to about 10⁻³, about 10⁻³ to about 10⁻²⁰, about 10⁻³ toabout 10⁻¹⁹, about 10⁻³ to about 10⁻¹⁸, about 10⁻³ to about 10⁻¹⁷, about10⁻³ to about 10⁻¹⁶, about 10⁻³ to about 10⁻¹⁵, about 10⁻³ to about10⁻¹⁴, about 10⁻³ to about 10⁻¹³, about 10⁻³ to about 10⁻¹², about 10⁻³to about 10⁻¹¹, about 10⁻³ to about 10⁻¹⁰, about 10⁻³ to about 10⁻⁹,about 10⁻³ to about 10⁻⁸, about 10⁻³ to about 10⁻⁷, about 10⁻³ to about10⁻⁶, about 10⁻³ to about 10⁻⁵, about 10⁻³ to about 10⁻⁴, about 10⁻⁴ toabout 10⁻²⁰, about 10⁻⁴ to about 10⁻¹⁹, about 10⁻⁴ to about 10⁻¹⁸, about10⁻⁴ to about 10⁻¹⁷, about 10⁻⁴ to about 10⁻¹⁶ about 10⁻⁴ to about10⁻¹⁵, about 10⁻⁴ to about 10⁻¹⁴, about 10⁻⁴ to about 10⁻¹³, about 10⁻⁴to about 10⁻¹², about 10⁻⁴ to about 10⁻¹¹, about 10⁻⁴ to about 10⁻¹⁰,about 10⁻⁴ to about 10⁻⁹, about 10⁻⁴ to about 10⁻⁸, about 10⁻⁴ to about10⁻⁷, about 10⁻⁴ to about 10⁻⁶, about 10⁻⁴ to about 10⁻⁵, about 10⁻⁵ toabout 10⁻²⁰, about 10⁻⁵ to about 10⁻¹⁹, about 10⁻⁵ to about 10⁻¹⁸, about10⁻⁵ to about 10⁻¹⁷, about 10⁻⁵ to about 10⁻¹⁶, about 10⁻⁵ to about10⁻¹⁵, about 10⁻⁵ to about 10⁻¹⁴, about 10⁻⁵ to about 10⁻¹³, about 10⁻⁵to about 10⁻¹², about 10⁻⁵ to about 10⁻¹¹, about 10⁻⁵ to about 10⁻¹⁰,about 10⁻⁵ to about 10⁻⁹, about 10⁻⁵ to about 10⁻⁸, about 10⁻⁵ to about10⁻⁷, about 10⁻⁵ to about 10⁻⁶, about 10⁻⁶ to about 10⁻²⁰, about 10⁻⁶ toabout 10⁻¹⁹, about 10⁻⁶ to about 10⁻¹⁸, about 10⁻⁶ to about 10⁻¹⁷, about10⁻⁶ to about 10⁻¹⁶, about 10⁻⁶ to about 10⁻¹⁵, about 10⁻⁶ to about10⁻¹⁴, about 10⁻⁶ to about 10⁻¹³, about 10⁻⁶ to about 10⁻¹² about 10⁻⁶to about 10⁻¹¹, about 10⁻⁶ to about 10⁻¹⁰, about 10⁻⁶ to about 10⁻⁹,about 10⁻⁶ to about 10⁻⁸, about 10⁻⁶ to about 10⁻⁷, about 10⁻⁷ to about10⁻²⁰, about 10⁻⁷ to about 10⁻¹⁹, about 10⁻⁷ to about 10⁻¹⁸, about 10⁻⁷to about 10⁻¹⁷, about 10⁻⁷ to about 10⁻¹⁶, about 10⁻⁷ to about 10⁻¹⁵,about 10⁻⁷ to about 10⁻¹⁴, about 10⁻⁷ to about 10⁻¹³, about 10⁻⁷ toabout 10⁻¹² about 10⁻⁷ to about 10⁻¹¹, about 10⁻⁷ to about 10⁻¹⁰, about10⁻⁷ to about 10⁻⁹, about 10⁻⁷ to about 10⁻⁸, about 10⁻⁸ to about 10⁻²⁰,about 10⁻⁸ to about 10⁻¹⁹, about 10⁻⁸ to about 10⁻¹⁸, about 10⁻⁸ toabout 10⁻¹⁷, about 10⁻⁸ to about 10⁻¹⁶, about 10⁻⁸ to about 10⁻¹⁵, about10⁻⁸ to about 10⁻¹⁴, about 10⁻⁸ to about 10⁻¹³, about 10⁻⁸ to about10⁻¹², about 10⁻⁸ to about 10⁻¹¹, about 10⁻⁸ to about 10⁻¹⁰, about 10⁻⁸to about 10⁻⁹, about 10⁻⁹ to about 10⁻²⁰, about 10⁻⁹ to about 10⁻¹⁹,about 10⁻⁹ to about 10⁻¹⁸, about 10⁻⁹ to about 10⁻¹⁷, about 10⁻⁹ toabout 10⁻¹⁶ about 10⁻⁹ to about 10⁻¹⁵, about 10⁻⁹ to about 10⁻¹⁴, about10⁻⁹ to about 10⁻¹³, about 10⁻⁹ to about 10⁻¹², about 10⁻⁹ to about10⁻¹¹, about 10⁻⁹ to about 10⁻¹⁰, about 10⁻¹⁰ to about 10⁻²⁰, about10⁻¹⁰ to about 10⁻¹⁹, about 10⁻¹⁰ to about 10⁻¹⁸, about 10⁻¹⁰ to about10⁻¹⁷, about 10⁻¹⁰ to about 10⁻¹⁶, about 10⁻¹⁰ to about 10⁻¹⁵, about10⁻¹⁰ to about 10⁻¹⁴, about 10⁻¹⁰ to about 10⁻¹³, about 10⁻¹⁰ to about10⁻¹², about 10⁻¹⁰ to about 1011, about 1011 to about 10⁻²⁰ about 10⁻¹¹to about 10⁻¹⁹, about 10⁻¹¹ to about 10⁻¹⁸, about 10⁻¹¹ to about 10⁻¹⁷,about 10⁻¹¹ to about 10⁻¹⁶, about 10⁻¹¹ to about 10⁻¹⁵, about 10⁻¹¹ toabout 10⁻¹⁴, about 10⁻¹¹ to about 10⁻¹³, about 1011 to about 10⁻¹²,about 10⁻¹² to about 10⁻²⁰, about 10⁻¹² to about 10⁻¹⁹ about 10⁻¹² toabout 10⁻¹⁸, about 10⁻¹² to about 10⁻¹⁷, about 10⁻¹² to about 10⁻¹⁶,about 10⁻¹² to about 10⁻¹⁵, about 10⁻¹² to about 10⁻¹⁴, about 10⁻¹² toabout 10⁻¹³, about 10⁻¹³ to about 10⁻²⁰, about 10⁻¹³ to about 10⁻¹⁹,about 1013 to about 1018, about 10⁻¹³ to about 10⁻¹⁷, about 10⁻¹³ toabout 10⁻¹⁶, about 10⁻¹³ to about 10⁻¹⁵, about 10⁻¹³ to about 10⁻¹⁴,about 10⁻¹⁴ to about 10⁻²⁰, about 10⁻¹⁴ to about 10⁻¹⁹, about 10⁻¹⁴ toabout 10⁻¹⁸, about 10⁻¹⁴ to about 10⁻¹⁷, about 10⁻¹⁴ to about 10⁻¹⁶,about 10⁻¹⁴ to about 1015, about 10⁻¹⁵ to about 10⁻²⁰ about 10⁻¹⁵ toabout 10⁻¹⁹, about 10⁻¹⁵ to about 10⁻¹⁸, about 10⁻¹⁵ to about 10⁻¹⁷,about 10⁻¹⁵ to about 10⁻¹⁶, about 10⁻¹⁶ to about 10⁻²⁰, about 10⁻¹⁶ toabout 10⁻¹⁹, about 10⁻¹⁶ to about 10⁻¹⁸, about 10⁻¹⁶ to about 10⁻¹⁷,about 1017 to about 10⁻²⁰, about 10⁻¹⁷ to about 10⁻¹⁹ about 10⁻¹⁷ toabout 10⁻¹⁸, about 10⁻¹⁸ to about 10⁻²⁰, about 10⁻¹⁸ to about 10⁻¹⁹, orabout 10⁻¹⁹ to about 10⁻²⁰.

In some embodiments, the step of continuously flowing the collectedfluid of step (d) into the second circuit system including a TFVF unitcan be performed such that the flux across the filter or one or morefilters in the TFVF unit is, e.g., about 10 LMH to about 50 LMH, about10 LMB to about 48 LMH, about 10 LMH to about 46 LMH, about 10 LMH toabout 44 LMH, about 10 LMH to about 42 LMH, about 10 LMH to about 40LMH, about 10 LMH to about 38 LMH, about 10 LMH to about 36 LMH, about10 LMH to about 34 LMH, about 10 LMH to about 32 LMH, about 10 LMH toabout 30 LMH, about 10 LMH to about 28 LMH, about 10 LMH to about 26LMH, about 10 LMH to about 24 LMH, about 10 LMH to about 22 LMH, about10 LMH to about 20 LMH, about 10 LMH to about 18 LMH, about 10 LMH toabout 16 LMH, about 10 LMH to about 15 LMH, about 15 LMH to about 50LMH, about 15 LMB to about 48 LMH, about 15 LMH to about 46 LMH, about15 LMH to about 44 LMH, about 15 LMH to about 42 LMH, about 15 LMH toabout 40 LMH, about 15 LMH to about 38 LMH, about 15 LMH to about 36LMH, about 15 LMH to about 34 LMH, about 15 LMH to about 32 LMH, about15 LMH to about 30 LMH, about 15 LMH to about 28 LMH, about 15 LMH toabout 26 LMH, about 15 LMH to about 24 LMH, about 15 LMH to about 22LMH, about 15 LMH to about 20 LMH, about 15 LMH to about 18 LMH, about20 LMH to about 50 LMH, about 20 LMB to about 48 LMH, about 20 LMH toabout 46 LMH, about 20 LMH to about 44 LMH, about 20 LMH to about 42LMH, about 20 LMH to about 40 LMH, about 20 LMH to about 38 LMH, about20 LMH to about 36 LMH, about 20 LMH to about 34 LMH, about 20 LMH toabout 32 LMH, about 20 LMH to about 30 LMH, about 20 LMH to about 28LMH, about 20 LMH to about 26 LMH, about 20 LMH to about 24 LMH, about20 LMH to about 22 LMH, about 22 LMH to about 50 LMH, about 22 LMB toabout 48 LMH, about 22 LMH to about 46 LMH, about 22 LMH to about 44LMH, about 22 LMH to about 42 LMH, about 22 LMH to about 40 LMH, about22 LMH to about 38 LMH, about 22 LMH to about 36 LMH, about 22 LMH toabout 34 LMH, about 22 LMH to about 32 LMH, about 22 LMH to about 30LMH, about 22 LMH to about 28 LMH, about 22 LMH to about 26 LMH, about22 LMH to about 24 LMH, about 24 LMH to about 50 LMH, about 24 LMB toabout 48 LMH, about 24 LMH to about 46 LMH, about 24 LMH to about 44LMH, about 24 LMH to about 42 LMH, about 24 LMH to about 40 LMH, about24 LMH to about 38 LMH, about 24 LMH to about 36 LMH, about 24 LMH toabout 34 LMH, about 24 LMH to about 32 LMH, about 24 LMH to about 30LMH, about 24 LMH to about 28 LMH, about 24 LMH to about 26 LMH, about26 LMH to about 50 LMH, about 26 LMB to about 48 LMH, about 26 LMH toabout 46 LMH, about 26 LMH to about 44 LMH, about 26 LMH to about 42LMH, about 26 LMH to about 40 LMH, about 26 LMH to about 38 LMH, about26 LMH to about 36 LMH, about 26 LMH to about 34 LMH, about 26 LMH toabout 32 LMH, about 26 LMH to about 30 LMH, about 26 LMH to about 28LMH, about 28 LMH to about 50 LMH, about 28 LMB to about 48 LMH, about28 LMH to about 46 LMH, about 28 LMH to about 44 LMH, about 28 LMH toabout 42 LMH, about 28 LMH to about 40 LMH, about 28 LMH to about 38LMH, about 28 LMH to about 36 LMH, about 28 LMH to about 34 LMH, about28 LMH to about 32 LMH, about 28 LMH to about 30 LMH, about 30 LMH toabout 50 LMH, about 30 LMB to about 48 LMH, about 30 LMH to about 46LMH, about 30 LMH to about 44 LMH, about 30 LMH to about 42 LMH, about30 LMH to about 40 LMH, about 30 LMH to about 38 LMH, about 30 LMH toabout 36 LMH, about 30 LMH to about 34 LMH, about 30 LMH to about 32LMH, about 32 LMH to about 50 LMH, about 32 LMB to about 48 LMH, about32 LMH to about 46 LMH, about 32 LMH to about 44 LMH, about 32 LMH toabout 42 LMH, about 32 LMH to about 40 LMH, about 32 LMH to about 38LMH, about 32 LMH to about 36 LMH, about 32 LMH to about 34 LMH, about34 LMH to about 50 LMH, about 34 LMB to about 48 LMH, about 34 LMH toabout 46 LMH, about 34 LMH to about 44 LMH, about 34 LMH to about 42LMH, about 34 LMH to about 40 LMH, about 34 LMH to about 38 LMH, about34 LMH to about 36 LMH, about 36 LMH to about 50 LMH, about 36 LMB toabout 48 LMH, about 36 LMH to about 46 LMH, about 36 LMH to about 44LMH, about 36 LMH to about 42 LMH, about 36 LMH to about 40 LMH, about36 LMH to about 38 LMH, about 38 LMH to about 50 LMH, about 38 LMB toabout 48 LMH, about 38 LMH to about 46 LMH, about 38 LMH to about 44LMH, about 38 LMH to about 42 LMH, about 38 LMH to about 40 LMH, about40 LMH to about 50 LMH, about 40 LMB to about 48 LMH, about 40 LMH toabout 46 LMH, about 40 LMH to about 44 LMH, about 40 LMH to about 42LMH, about 42 LMH to about 50 LMH, about 42 LMB to about 48 LMH, about42 LMH to about 46 LMH, about 42 LMH to about 44 LMH, about 44 LMH toabout 50 LMH, about 44 LMB to about 48 LMH, about 44 LMH to about 46LMH, about 46 LMH to about 50 LMH, about 46 LMB to about 48 LMH, orabout 48 LMH to about 50 LMH.

In some embodiments, the step of continuously flowing the collectedfluid from step (d) into the second circuit system including a TFVF unitcan be performed such that the pressure across the filter or the one ormore filters in the TFVF unit, or the pressure in the second systemincluding the TFVF is, e.g., about 2 psi to about 40 psi, about 2 psi toabout 38 psi, about 2 psi to about 36 psi, about 2 psi to about 34 psi,about 2 psi to about 32 psi, about 2 psi to about 30 psi, about 2 psi toabout 28 psi, about 2 psi to about 26 psi, about 2 psi to about 24 psi,about 2 psi to about 22 psi, about 2 psi to about 20 psi, about 2 psi toabout 18 psi, about 2 psi to about 16 psi, about 2 psi to about 14 psi,about 2 psi to about 12 psi, about 2 psi to about 10 psi, about 2 psi toabout 8 psi, about 2 psi to about 6 psi, about 2 psi to about 4 psi,about 4 psi to about 40 psi, about 4 psi to about 38 psi, about 4 psi toabout 36 psi, about 4 psi to about 34 psi, about 4 psi to about 32 psi,about 4 psi to about 30 psi, about 4 psi to about 28 psi, about 4 psi toabout 26 psi, about 4 psi to about 24 psi, about 4 psi to about 22 psi,about 4 psi to about 20 psi, about 4 psi to about 18 psi, about 4 psi toabout 16 psi, about 4 psi to about 14 psi, about 4 psi to about 12 psi,about 4 psi to about 10 psi, about 4 psi to about 8 psi, about 4 psi toabout 6 psi, about 6 psi to about 40 psi, about 6 psi to about 38 psi,about 6 psi to about 36 psi, about 6 psi to about 34 psi, about 6 psi toabout 32 psi, about 6 psi to about 30 psi, about 6 psi to about 28 psi,about 6 psi to about 26 psi, about 6 psi to about 24 psi, about 6 psi toabout 22 psi, about 6 psi to about 20 psi, about 6 psi to about 18 psi,about 6 psi to about 16 psi, about 6 psi to about 14 psi, about 6 psi toabout 12 psi, about 6 psi to about 10 psi, about 6 psi to about 8 psi,about 8 psi to about 40 psi, about 8 psi to about 38 psi, about 8 psi toabout 36 psi, about 8 psi to about 34 psi, about 8 psi to about 32 psi,about 8 psi to about 30 psi, about 8 psi to about 28 psi, about 8 psi toabout 26 psi, about 8 psi to about 24 psi, about 8 psi to about 22 psi,about 8 psi to about 20 psi, about 8 psi to about 18 psi, about 8 psi toabout 16 psi, about 8 psi to about 14 psi, about 8 psi to about 12 psi,about 8 psi to about 10 psi, about 10 psi to about 40 psi, about 10 psito about 38 psi, about 10 psi to about 36 psi, about 10 psi to about 34psi, about 10 psi to about 32 psi, about 10 psi to about 30 psi, about10 psi to about 28 psi, about 10 psi to about 26 psi, about 10 psi toabout 24 psi, about 10 psi to about 22 psi, about 10 psi to about 20psi, about 10 psi to about 18 psi, about 10 psi to about 16 psi, about10 psi to about 14 psi, about 10 psi to about 12 psi, about 12 psi toabout 40 psi, about 12 psi to about 38 psi, about 12 psi to about 36psi, about 12 psi to about 34 psi, about 12 psi to about 32 psi, about12 psi to about 30 psi, about 12 psi to about 28 psi, about 12 psi toabout 26 psi, about 12 psi to about 24 psi, about 12 psi to about 22psi, about 12 psi to about 20 psi, about 12 psi to about 18 psi, about12 psi to about 16 psi, about 12 psi to about 14 psi, about 14 psi toabout 40 psi, about 14 psi to about 38 psi, about 14 psi to about 36psi, about 14 psi to about 34 psi, about 14 psi to about 32 psi, about14 psi to about 30 psi, about 14 psi to about 28 psi, about 14 psi toabout 26 psi, about 14 psi to about 24 psi, about 14 psi to about 22psi, about 14 psi to about 20 psi, about 14 psi to about 18 psi, about14 psi to about 16 psi, about 16 psi to about 40 psi, about 16 psi toabout 38 psi, about 16 psi to about 36 psi, about 16 psi to about 34psi, about 16 psi to about 32 psi, about 16 psi to about 30 psi, about16 psi to about 28 psi, about 16 psi to about 26 psi, about 16 psi toabout 24 psi, about 16 psi to about 22 psi, about 16 psi to about 20psi, about 16 psi to about 18 psi, about 18 psi to about 40 psi, about18 psi to about 38 psi, about 18 psi to about 36 psi, about 18 psi toabout 34 psi, about 18 psi to about 32 psi, about 18 psi to about 30psi, about 18 psi to about 28 psi, about 18 psi to about 26 psi, about18 psi to about 24 psi, about 18 psi to about 22 psi, about 18 psi toabout 20 psi, about 20 psi to about 40 psi, about 20 psi to about 38psi, about 20 psi to about 36 psi, about 20 psi to about 34 psi, about20 psi to about 32 psi, about 20 psi to about 30 psi, about 20 psi toabout 28 psi, about 20 psi to about 26 psi, about 20 psi to about 24psi, about 20 psi to about 22 psi, about 22 psi to about 40 psi, about22 psi to about 38 psi, about 22 psi to about 36 psi, about 22 psi toabout 34 psi, about 22 psi to about 32 psi, about 22 psi to about 30psi, about 22 psi to about 28 psi, about 22 psi to about 26 psi, about22 psi to about 24 psi, about 24 psi to about 40 psi, about 24 psi toabout 38 psi, about 24 psi to about 36 psi, about 24 psi to about 34psi, about 24 psi to about 32 psi, about 24 psi to about 30 psi, about24 psi to about 28 psi, about 24 psi to about 26 psi, about 26 psi toabout 40 psi, about 26 psi to about 38 psi, about 26 psi to about 36psi, about 26 psi to about 34 psi, about 26 psi to about 32 psi, about26 psi to about 30 psi, about 26 psi to about 28 psi, about 28 psi toabout 40 psi, about 28 psi to about 38 psi, about 28 psi to about 36psi, about 28 psi to about 34 psi, about 28 psi to about 32 psi, about28 psi to about 30 psi, about 30 psi to about 40 psi, about 30 psi toabout 38 psi, about 30 psi to about 36 psi, about 30 psi to about 34psi, about 30 psi to about 32 psi, about 32 psi to about 40 psi, about32 psi to about 38 psi, about 32 psi to about 36 psi, about 32 psi toabout 34 psi, about 34 psi to about 40 psi, about 34 psi to about 38psi, about 34 psi to about 36 psi, about 36 psi to about 40 psi, about36 psi to about 38 psi, or about 38 psi to about 40 psi.

Methods of Processing a Fluid Including a Recombinant TherapeuticProtein that Include the Use of a Circuit System Including a TFVF Unit

Also provided are methods of processing a fluid including a recombinanttherapeutic protein that include: (a) providing a circuit systemincluding (i) a TFVF unit having first and second inlets, and (ii) aconduit in fluid communication between the first and second inlets ofthe TFVF unit, including at least one port for flowing fluid into or outof, or both, of the system, where the system is configured such thatfluid can be flowed through the conduit and the TFVF unit, and filtrateincluding the recombinant therapeutic protein can be collected from theTFVF unit; (b) continuously flowing a fluid including a recombinanttherapeutic protein into the circuit system through one of the at leastone port, and collecting filtrate including the recombinant therapeuticprotein from the TFVF unit for a period of time. As can be appreciatedby those skilled in the art, some embodiments of these methods canfurther include recirculating any fluid including the recombinanttherapeutic protein in the conduit of the circuit system including aTFVF unit that did not pass through the one or more tangential virusfilters in the TFVF unit (e.g., acts as a buffer when a new volume of afluid including the recombinant therapeutic protein is flowed into theconduit of the circuit system including a TFVF unit).

In some embodiments, the TFVF unit includes one, two, three, four, five,six, seven, eight, nine, ten, eleven, or twelve tangential virusfilters. In some examples of these methods, the TFVF unit can includeone or more tangential virus filter(s) having a surface area of about0.05 cm² to about 100 m² (or any of the subranges of this rangedescribed herein).

In some embodiments, the TFVF unit includes one or more tangential virusfilters having a pore size of about 5 nm to about 220 nm (or any of thesubranges of this range described herein). A variety of virus filtersthat can be used in a TFVF unit are known in the art. For example, virusfilters that can be used in a TFVF unit are commercially available fromAsahi Kasei (Tokyo, Japan). In some embodiments, the virus filter is aPlanova™ filter (e.g, a Planova™ 15N, a Planova™ 20N, a Planova™ 35N, aPlanova™ 75N). In some embodiments, the virus filter is a Planova™ BioEXfilter. The virus filters that can be used in a TFVF unit can include,e.g., hollow fiber membranes.

The conduit can be simple tubing, e.g., biocompatible tubing.Non-limiting examples of useful tubing include silicone rubber,polyurethane, polydioxanone (PDO), polyhydroxyalkanoate,polyhydroxybutyrate, poly(glycerol sebacate), polyglycolide,polylactide, polycaprolactone, or polyanhydride, or copolymers orderivatives including these and/or other polymers. Alternatively or inaddition, the conduit can include polyvinyl chloride. The conduit canbe, e.g., weldable transfer tubing.

In some embodiments, the internal diameter of the conduit in the circuitsystem can be about 5 mm to about 50 mm (e.g., or any of the subrangesthereof described herein). In some embodiments, the conduit in thecircuit system has a total length of about 1.0 cm to about 75 cm (or anyof the subranges of this range described herein). Additional examples ofconduits and properties of conduits that can be used in the secondcircuit system are well-known by those in the art.

In some examples, the circuit system includes one, two, three, four, orfive ports. In some examples, the at least one port in the circuitsystem can be a valve. In other examples, the at least one port in thecircuit system can be an injection port or can have a ribbed threading.The at least one port in the circuit system can be any type of portcommonly known in the art. In some examples, the circuit system canfurther include a pump (e.g., a low-turbulence pump, e.g., a peristalticpump) disposed in the conduit. In some embodiments, the circuit systemdoes not include a holding tank.

In some examples, step (b) includes continuously flowing (e.g.,unidirectionally or bidirectionally flowing) a fluid including arecombinant therapeutic protein into the circuit system at a rate ofbetween about 0.05 mL/minute to about 25 mL/minute (or any of thesubranges of this range described herein). In some examples, thefiltrate collected from the TFVF unit in step (b) includes aconcentration of recombinant therapeutic protein of about 0.1 mg/mL toabout 100 mg/mL (or any of the subranges of this range describedherein). In some examples of these methods, the period of time can beabout 5 minutes to about 12 hours (or any of the subranges of this rangedescribed herein).

In some examples of these methods, the period of time can be, e.g.,about 12 hours to about 72 hours (or any of the subranges of this rangedescribed herein).

In some embodiments, the fluid that is continuously flowed into thecircuit system including the TFVF unit through one of the at least oneport can include a total virus titer of about 10⁻¹ to about 10⁻²⁰ (orany of the subranges of this range described herein).

In some embodiments, the step of continuously flowing the fluid throughthe circuit system including a TFVF unit can be performed such that theflux across the filter or one or more filters in the TFVF unit is, e.g.,about 10 LMH to about 50 LMH (or any of the subranges of this rangedescribed herein).

In some embodiments, the step of continuously flowing the fluid throughthe circuit system including a TFVF unit can be performed such that thepressure across the filter or the one or more filters in the TFVF unit,or the pressure in the system including the TFVF is, e.g., about 2 psito about 40 psi (or any of the subranges of this range describedherein).

In some embodiments, the method can further include performing one ormore unit operations on a fluid including the recombinant protein.Non-limiting examples of these unit operations include: capturing arecombinant therapeutic protein, purifying a recombinant therapeuticprotein, polishing a recombinant therapeutic protein, viralinactivation, viral filtration, adjusting the ionic concentration and/orpH of a fluid containing the recombinant therapeutic protein, filtrationof a fluid containing the recombinant therapeutic protein, precipitationand flocculation, aqueous two-phase extraction, lyophilization, andcrystallization. Non-limiting aspects of each of these exemplary unitoperations are described herein.

Integrated and Continuous Processes for Manufacturing a TherapeuticProtein Drug Substance

Provided herein are integrated and continuous processes formanufacturing a therapeutic protein drug substance. These processesinclude providing a liquid culture medium containing a recombinanttherapeutic protein that is substantially free of cells, where theliquid culture medium is fed into a first multi-column chromatographysystem (MCCS1); capturing the recombinant therapeutic protein in theliquid culture medium using the MCCS1, where the eluate of the MCCS1containing the recombinant therapeutic protein is continuously fed intoa second multi-column chromatography system (MCCS2); purifying andpolishing the recombinant therapeutic protein using the MCCS2, where theeluate from the MCCS2 is a fluid including the recombinant therapeuticprotein; processing the fluid including the recombinant therapeuticprotein using any of the methods described herein, where the process isintegrated and runs continuously from the liquid culture medium to thecollected fluid or filtrate including the recombinant therapeuticprotein, where the collected fluid or filtrate is the therapeuticprotein drug substance. Examples of systems that can be used to performthese processes are shown in FIGS. 1 and 2 (where asterisks indicatewhere one or both of a circuit system including tangential flowfiltration unit and a tangential flow virus filtration unit canoptionally be introduced into the system). For example, one or both of acircuit system including tangential flow filtration unit and atangential flow viral filtration unit can be used before the performanceof a first unit operation, between the performance of a first and secondunit operation, between the performance of a second and a third unitoperation, between the performance of a third and a fourth unitoperation, between the performance of a fourth and a fifth unitoperation, between the performance of a fifth and a sixth unitoperation, between the performance of a sixth and a seventh unitoperation, between the performance of a seventh and an eighth unitoperation, and/or between an eighth and a ninth unit operation.

The processes described herein provide continuous and time-efficientproduction of a therapeutic drug substance from a liquid culture medium.For example, the elapsed time between feeding a fluid (e.g., a liquidculture medium) containing a therapeutic protein into the first MCCS andeluting a therapeutic protein drug substance (containing the therapeuticprotein) from the second MCCS can be, e.g., between about 4 hours andabout 48 hours, inclusive, e.g., between about 4 hours and about 40hours, between about 4 hours and about 35 hours, between about 4 hoursand about 30 hours, between about 4 hours and about 28 hours, betweenabout 4 hours and about 26 hours, between about 4 hours and about 24hours, between about 4 hours and about 22 hours, between about 4 hoursand about 20 hours, between about 4 hours and about 18 hours, betweenabout 4 hours and about 16 hours, between about 4 hours and about 14hours, between about 4 hours and about 12 hours, between about 6 hoursand about 12 hours, between about 8 hours and about 12 hours, betweenabout 6 hours and about 20 hours, between about 6 hours and about 18hours, between about 6 hours and about 14 hours, between about 8 hoursand about 16 hours, between about 8 hours and about 14 hours, betweenabout 8 hours and about 12 hours, between about 10 hours and 20 hours,between about 10 hours and 18 hours, between about 10 hours and 16hours, between about 10 hours and 14 hours, between about 12 hours andabout 14 hours, between about 10 hours and about 40 hours, between about10 hours and about 35 hours, between about 10 hours and about 30 hours,between about 10 hours and about 25 hours, between about 15 hours andabout 40 hours, between about 15 hours and about 35 hours, between about15 hours and about 30 hours, between about 20 hours and about 40 hours,between about 20 hours and about 35 hours, or between about 20 hours andabout 30 hours, inclusive. In other examples, the elapsed time betweenfeeding the fluid (e.g., a liquid culture medium) containing atherapeutic protein into the MCCS1 and eluting a therapeutic proteindrug substance (containing the therapeutic protein) from the MCCS2 is,e.g., greater than about 4 hours and less than about 40 hours,inclusive, e.g., greater than about 4 hours and less than about 39hours, about 38 hours, about 37 hours, about 36 hours, about 35 hours,about 34 hours, about 33 hours, about 32 hours, about 31 hours, about 30hours, about 29 hours, about 28 hours, about 27 hours, about 26 hours,about 25 hours, about 24 hours, about 23 hours, about 22 hours, about 21hours, about 20 hours, about 19 hours, about 18 hours, about 17 hours,about 16 hours, about 15 hours, about 14 hours, about 13 hours, about 12hours, about 11 hours, about 10 hours, about 9 hours, about 8 hours,about 7 hours, about 6 hours, about 5 hours, or about 4.5 hours,inclusive.

Some exemplary processes do not utilize a holding step (e.g., do not usea reservoir (e.g., break tank) in the entire process). Others may use amaximum of 1, 2, 3, 4, or 5 reservoir(s) (e.g., break tank(s)) in theentire process. Any of the processes described herein can utilize amaximum of 1, 2, 3, 4, or 5 reservoir(s) (e.g., break tank(s)) in theentire process, where each break tank only holds a therapeutic proteinfor a total time period of, e.g., between about 5 minutes and less thanabout 6 hours, inclusive, e.g., between about 5 minutes and about 5hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, orabout 30 minutes, inclusive. Any of the reservoir(s) (e.g., breaktank(s)) used in the processes described herein can have a capacity thatis, e.g., between 1 mL and about 10,000 L, about 9,500 L, about 9,000 L,about 8,500 L, about 8,000 L, about 7,500 L, about 7,000 L, about 6,500L, about 6,000 L, about 5,500 L, about 5,000 L, about 4,500 L, about4,000 L, about 3,500 L, about 3,000 L, about 2,500 L, about 2,000 L,about 1,500 L, about 1,000 L, about 950 mL, about 900 mL, about 850 mL,about 800 mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL,about 550 mL, about 500 mL, about 450 mL, about 400 mL, about 350 mL,about 300 mL, about 280 mL, about 260 mL, about 240 mL, about 220 mL,about 200 mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL,about 100 mL, about 80 mL, about 60 mL, about 40 mL, about 20 mL, orabout 10 mL (inclusive); between about 5 mL and about 10,000 L, about9,500 L, about 9,000 L, about 8,500 L, about 8,000 L, about 7,500 L,about 7,000 L, about 6,500 L, about 6,000 L, about 5,500 L, about 5,000L, about 4,500 L, about 4,000 L, about 3,500 L, about 3,000 L, about2,500 L, about 2,000 L, about 1,500 L, about 1,000 L, about 950 mL,about 900 mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL,about 650 mL, about 600 mL, about 550 mL, about 500 mL, about 450 mL,about 400 mL, about 350 mL, about 300 mL, about 280 mL, about 260 mL,about 240 mL, about 220 mL, about 200 mL, about 180 mL, about 160 mL,about 140 mL, about 120 mL, about 100 mL, about 80 mL, about 60 mL,about 40 mL, about 20 mL, or about 10 mL (inclusive); between about 10mL and about 10,000 L, about 9,500 L, about 9,000 L, about 8,500 L,about 8,000 L, about 7,500 L, about 7,000 L, about 6,500 L, about 6,000L, about 5,500 L, about 5,000 L, about 4,500 L, about 4,000 L, about3,500 L, about 3,000 L, about 2,500 L, about 2,000 L, about 1,500 L,about 1,000 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL,about 750 mL, about 700 mL, about 650 mL, about 600 mL, about 550 mL,about 500 mL, about 450 mL, about 400 mL, about 350 mL, about 300 mL,about 280 mL, about 260 mL, about 240 mL, about 220 mL, about 200 mL,about 180 mL, about 160 mL, about 140 mL, about 120 mL, about 100 mL,about 80 mL, about 60 mL, about 40 mL, or about 20 mL (inclusive);between about 20 mL and about 10,000 L, about 9,500 L, about 9,000 L,about 8,500 L, about 8,000 L, about 7,500 L, about 7,000 L, about 6,500L, about 6,000 L, about 5,500 L, about 5,000 L, about 4,500 L, about4,000 L, about 3,500 L, about 3,000 L, about 2,500 L, about 2,000 L,about 1,500 L, about 1,000 L, about 950 mL, about 900 mL, about 850 mL,about 800 mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL,about 550 mL, about 500 mL, about 450 mL, about 400 mL, about 350 mL,about 300 mL, about 280 mL, about 260 mL, about 240 mL, about 220 mL,about 200 mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL,about 100 mL, about 80 mL, about 60 mL, or about 40 mL (inclusive);between about 40 mL and about 10,000 L, about 9,500 L, about 9,000 L,about 8,500 L, about 8,000 L, about 7,500 L, about 7,000 L, about 6,500L, about 6,000 L, about 5,500 L, about 5,000 L, about 4,500 L, about4,000 L, about 3,500 L, about 3,000 L, about 2,500 L, about 2,000 L,about 1,500 L, about 1,000 L, about 950 mL, about 900 mL, about 850 mL,about 800 mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL,about 550 mL, about 500 mL, about 450 mL, about 400 mL, about 350 mL,about 300 mL, about 280 mL, about 260 mL, about 240 mL, about 220 mL,about 200 mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL,about 100 mL, about 80 mL, or about 60 mL (inclusive); between about 60mL and about 10,000 L, about 9,500 L, about 9,000 L, about 8,500 L,about 8,000 L, about 7,500 L, about 7,000 L, about 6,500 L, about 6,000L, about 5,500 L, about 5,000 L, about 4,500 L, about 4,000 L, about3,500 L, about 3,000 L, about 2,500 L, about 2,000 L, about 1,500 L,about 1,000 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL,about 750 mL, about 700 mL, about 650 mL, about 600 mL, about 550 mL,about 500 mL, about 450 mL, about 400 mL, about 350 mL, about 300 mL,about 280 mL, about 260 mL, about 240 mL, about 220 mL, about 200 mL,about 180 mL, about 160 mL, about 140 mL, about 120 mL, about 100 mL, orabout 80 mL (inclusive); between about 80 mL and about 10,000 L, about9,500 L, about 9,000 L, about 8,500 L, about 8,000 L, about 7,500 L,about 7,000 L, about 6,500 L, about 6,000 L, about 5,500 L, about 5,000L, about 4,500 L, about 4,000 L, about 3,500 L, about 3,000 L, about2,500 L, about 2,000 L, about 1,500 L, about 1,000 L, about 950 mL,about 900 mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL,about 650 mL, about 600 mL, about 550 mL, about 500 mL, about 450 mL,about 400 mL, about 350 mL, about 300 mL, about 280 mL, about 260 mL,about 240 mL, about 220 mL, about 200 mL, about 180 mL, about 160 mL,about 140 mL, about 120 mL, or about 100 mL (inclusive); between about100 mL and about 10,000 L, about 9,500 L, about 9,000 L, about 8,500 L,about 8,000 L, about 7,500 L, about 7,000 L, about 6,500 L, about 6,000L, about 5,500 L, about 5,000 L, about 4,500 L, about 4,000 L, about3,500 L, about 3,000 L, about 2,500 L, about 2,000 L, about 1,500 L,about 1,000 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL,about 750 mL, about 700 mL, about 650 mL, about 600 mL, about 550 mL,about 500 mL, about 450 mL, about 400 mL, about 350 mL, about 300 mL,about 280 mL, about 260 mL, about 240 mL, about 220 mL, about 200 mL,about 180 mL, about 160 mL, about 140 mL, or about 120 mL (inclusive);between about 120 mL and about 10,000 L, about 9,500 L, about 9,000 L,about 8,500 L, about 8,000 L, about 7,500 L, about 7,000 L, about 6,500L, about 6,000 L, about 5,500 L, about 5,000 L, about 4,500 L, about4,000 L, about 3,500 L, about 3,000 L, about 2,500 L, about 2,000 L,about 1,500 L, about 1,000 L, about 950 mL, about 900 mL, about 850 mL,about 800 mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL,about 550 mL, about 500 mL, about 450 mL, about 400 mL, about 350 mL,about 300 mL, about 280 mL, about 260 mL, about 240 mL, about 220 mL,about 200 mL, about 180 mL, about 160 mL, or about 140 mL (inclusive);between about 140 mL and about 10,000 L, about 9,500 L, about 9,000 L,about 8,500 L, about 8,000 L, about 7,500 L, about 7,000 L, about 6,500L, about 6,000 L, about 5,500 L, about 5,000 L, about 4,500 L, about4,000 L, about 3,500 L, about 3,000 L, about 2,500 L, about 2,000 L,about 1,500 L, about 1,000 L, about 950 mL, about 900 mL, about 850 mL,about 800 mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL,about 550 mL, about 500 mL, about 450 mL, about 400 mL, about 350 mL,about 300 mL, about 280 mL, about 260 mL, about 240 mL, about 220 mL,about 200 mL, about 180 mL, or about 160 mL (inclusive); between about160 mL and about 10,000 L, about 9,500 L, about 9,000 L, about 8,500 L,about 8,000 L, about 7,500 L, about 7,000 L, about 6,500 L, about 6,000L, about 5,500 L, about 5,000 L, about 4,500 L, about 4,000 L, about3,500 L, about 3,000 L, about 2,500 L, about 2,000 L, about 1,500 L,about 1,000 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL,about 750 mL, about 700 mL, about 650 mL, about 600 mL, about 550 mL,about 500 mL, about 450 mL, about 400 mL, about 350 mL, about 300 mL,about 280 mL, about 260 mL, about 240 mL, about 220 mL, about 200 mL, orabout 180 mL (inclusive); between about 180 mL and about 10,000 L, about9,500 L, about 9,000 L, about 8,500 L, about 8,000 L, about 7,500 L,about 7,000 L, about 6,500 L, about 6,000 L, about 5,500 L, about 5,000L, about 4,500 L, about 4,000 L, about 3,500 L, about 3,000 L, about2,500 L, about 2,000 L, about 1,500 L, about 1,000 L, about 950 mL,about 900 mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL,about 650 mL, about 600 mL, about 550 mL, about 500 mL, about 450 mL,about 400 mL, about 350 mL, about 300 mL, about 280 mL, about 260 mL,about 240 mL, about 220 mL, or about 200 mL (inclusive); between about200 mL and about 10,000 L, about 9,500 L, about 9,000 L, about 8,500 L,about 8,000 L, about 7,500 L, about 7,000 L, about 6,500 L, about 6,000L, about 5,500 L, about 5,000 L, about 4,500 L, about 4,000 L, about3,500 L, about 3,000 L, about 2,500 L, about 2,000 L, about 1,500 L,about 1,000 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL,about 750 mL, about 700 mL, about 650 mL, about 600 mL, about 550 mL,about 500 mL, about 450 mL, about 400 mL, about 350 mL, about 300 mL,about 280 mL, about 260 mL, about 240 mL, or about 220 mL (inclusive);between about 220 mL and about 10,000 L, about 9,500 L, about 9,000 L,about 8,500 L, about 8,000 L, about 7,500 L, about 7,000 L, about 6,500L, about 6,000 L, about 5,500 L, about 5,000 L, about 4,500 L, about4,000 L, about 3,500 L, about 3,000 L, about 2,500 L, about 2,000 L,about 1,500 L, about 1,000 L, about 950 mL, about 900 mL, about 850 mL,about 800 mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL,about 550 mL, about 500 mL, about 450 mL, about 400 mL, about 350 mL,about 300 mL, about 280 mL, about 260 mL, or about 240 mL (inclusive);between about 240 mL and about 10,000 L, about 9,500 L, about 9,000 L,about 8,500 L, about 8,000 L, about 7,500 L, about 7,000 L, about 6,500L, about 6,000 L, about 5,500 L, about 5,000 L, about 4,500 L, about4,000 L, about 3,500 L, about 3,000 L, about 2,500 L, about 2,000 L,about 1,500 L, about 1,000 L, about 950 mL, about 900 mL, about 850 mL,about 800 mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL,about 550 mL, about 500 mL, about 450 mL, about 400 mL, about 350 mL,about 300 mL, about 280 mL, or about 260 mL (inclusive); between about260 mL and about 10,000 L, about 9,500 L, about 9,000 L, about 8,500 L,about 8,000 L, about 7,500 L, about 7,000 L, about 6,500 L, about 6,000L, about 5,500 L, about 5,000 L, about 4,500 L, about 4,000 L, about3,500 L, about 3,000 L, about 2,500 L, about 2,000 L, about 1,500 L,about 1,000 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL,about 750 mL, about 700 mL, about 650 mL, about 600 mL, about 550 mL,about 500 mL, about 450 mL, about 400 mL, about 350 mL, about 300 mL, orabout 280 mL (inclusive); between about 280 mL and about 10,000 L, about9,500 L, about 9,000 L, about 8,500 L, about 8,000 L, about 7,500 L,about 7,000 L, about 6,500 L, about 6,000 L, about 5,500 L, about 5,000L, about 4,500 L, about 4,000 L, about 3,500 L, about 3,000 L, about2,500 L, about 2,000 L, about 1,500 L, about 1,000 L, about 950 mL,about 900 mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL,about 650 mL, about 600 mL, about 550 mL, about 500 mL, about 450 mL,about 400 mL, about 350 mL, or about 300 mL (inclusive); between about300 mL and about 10,000 L, about 9,500 L, about 9,000 L, about 8,500 L,about 8,000 L, about 7,500 L, about 7,000 L, about 6,500 L, about 6,000L, about 5,500 L, about 5,000 L, about 4,500 L, about 4,000 L, about3,500 L, about 3,000 L, about 2,500 L, about 2,000 L, about 1,500 L,about 1,000 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL,about 750 mL, about 700 mL, about 650 mL, about 600 mL, about 550 mL,about 500 mL, about 450 mL, about 400 mL, or about 350 mL (inclusive);between about 350 mL and about 10,000 L, about 9,500 L, about 9,000 L,about 8,500 L, about 8,000 L, about 7,500 L, about 7,000 L, about 6,500L, about 6,000 L, about 5,500 L, about 5,000 L, about 4,500 L, about4,000 L, about 3,500 L, about 3,000 L, about 2,500 L, about 2,000 L,about 1,500 L, about 1,000 L, about 950 mL, about 900 mL, about 850 mL,about 800 mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL,about 550 mL, about 500 mL, about 450 mL, or about 400 mL (inclusive);between about 400 mL and about 10,000 L, about 9,500 L, about 9,000 L,about 8,500 L, about 8,000 L, about 7,500 L, about 7,000 L, about 6,500L, about 6,000 L, about 5,500 L, about 5,000 L, about 4,500 L, about4,000 L, about 3,500 L, about 3,000 L, about 2,500 L, about 2,000 L,about 1,500 L, about 1,000 L, about 950 mL, about 900 mL, about 850 mL,about 800 mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL,about 550 mL, about 500 mL, or about 450 mL (inclusive); between about450 mL and about 10,000 L, about 9,500 L, about 9,000 L, about 8,500 L,about 8,000 L, about 7,500 L, about 7,000 L, about 6,500 L, about 6,000L, about 5,500 L, about 5,000 L, about 4,500 L, about 4,000 L, about3,500 L, about 3,000 L, about 2,500 L, about 2,000 L, about 1,500 L,about 1,000 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL,about 750 mL, about 700 mL, about 650 mL, about 600 mL, about 550 mL, orabout 500 mL (inclusive); between about 500 mL and about 10,000 L, about9,500 L, about 9,000 L, about 8,500 L, about 8,000 L, about 7,500 L,about 7,000 L, about 6,500 L, about 6,000 L, about 5,500 L, about 5,000L, about 4,500 L, about 4,000 L, about 3,500 L, about 3,000 L, about2,500 L, about 2,000 L, about 1,500 L, about 1,000 L, about 950 mL,about 900 mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL,about 650 mL, about 600 mL, or about 550 mL (inclusive); between about550 mL and about 10,000 L, about 9,500 L, about 9,000 L, about 8,500 L,about 8,000 L, about 7,500 L, about 7,000 L, about 6,500 L, about 6,000L, about 5,500 L, about 5,000 L, about 4,500 L, about 4,000 L, about3,500 L, about 3,000 L, about 2,500 L, about 2,000 L, about 1,500 L,about 1,000 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL,about 750 mL, about 700 mL, about 650 mL, or about 600 mL (inclusive);between about 600 mL and about 10,000 L, about 9,500 L, about 9,000 L,about 8,500 L, about 8,000 L, about 7,500 L, about 7,000 L, about 6,500L, about 6,000 L, about 5,500 L, about 5,000 L, about 4,500 L, about4,000 L, about 3,500 L, about 3,000 L, about 2,500 L, about 2,000 L,about 1,500 L, about 1,000 L, about 950 mL, about 900 mL, about 850 mL,about 800 mL, about 750 mL, about 700 mL, or about 650 mL (inclusive);between about 650 mL and about 10,000 L, about 9,500 L, about 9,000 L,about 8,500 L, about 8,000 L, about 7,500 L, about 7,000 L, about 6,500L, about 6,000 L, about 5,500 L, about 5,000 L, about 4,500 L, about4,000 L, about 3,500 L, about 3,000 L, about 2,500 L, about 2,000 L,about 1,500 L, about 1,000 L, about 950 mL, about 900 mL, about 850 mL,about 800 mL, about 750 mL, or about 700 mL (inclusive); between about700 mL and about 10,000 L, about 9,500 L, about 9,000 L, about 8,500 L,about 8,000 L, about 7,500 L, about 7,000 L, about 6,500 L, about 6,000L, about 5,500 L, about 5,000 L, about 4,500 L, about 4,000 L, about3,500 L, about 3,000 L, about 2,500 L, about 2,000 L, about 1,500 L,about 1,000 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL,or about 750 mL (inclusive); between about 750 mL and about 10,000 L,about 9,500 L, about 9,000 L, about 8,500 L, about 8,000 L, about 7,500L, about 7,000 L, about 6,500 L, about 6,000 L, about 5,500 L, about5,000 L, about 4,500 L, about 4,000 L, about 3,500 L, about 3,000 L,about 2,500 L, about 2,000 L, about 1,500 L, about 1,000 L, about 950mL, about 900 mL, about 850 mL, or about 800 mL (inclusive); betweenabout 800 mL and about 10,000 L, about 9,500 L, about 9,000 L, about8,500 L, about 8,000 L, about 7,500 L, about 7,000 L, about 6,500 L,about 6,000 L, about 5,500 L, about 5,000 L, about 4,500 L, about 4,000L, about 3,500 L, about 3,000 L, about 2,500 L, about 2,000 L, about1,500 L, about 1,000 L, about 950 mL, about 900 mL, or about 850 mL(inclusive); between about 850 mL and about 10,000 L, about 9,500 L,about 9,000 L, about 8,500 L, about 8,000 L, about 7,500 L, about 7,000L, about 6,500 L, about 6,000 L, about 5,500 L, about 5,000 L, about4,500 L, about 4,000 L, about 3,500 L, about 3,000 L, about 2,500 L,about 2,000 L, about 1,500 L, about 1,000 L, about 950 mL, or about 900mL (inclusive); between about 900 mL and about 10,000 L, about 9,500 L,about 9,000 L, about 8,500 L, about 8,000 L, about 7,500 L, about 7,000L, about 6,500 L, about 6,000 L, about 5,500 L, about 5,000 L, about4,500 L, about 4,000 L, about 3,500 L, about 3,000 L, about 2,500 L,about 2,000 L, about 1,500 L, about 1,000 L, or about 950 mL(inclusive); between about 950 mL and about 10,000 L, about 9,500 L,about 9,000 L, about 8,500 L, about 8,000 L, about 7,500 L, about 7,000L, about 6,500 L, about 6,000 L, about 5,500 L, about 5,000 L, about4,500 L, about 4,000 L, about 3,500 L, about 3,000 L, about 2,500 L,about 2,000 L, about 1,500 L, or about 1,000 L (inclusive); betweenabout 1,000 L and about 10,000 L, about 9,500 L, about 9,000 L, about8,500 L, about 8,000 L, about 7,500 L, about 7,000 L, about 6,500 L,about 6,000 L, about 5,500 L, about 5,000 L, about 4,500 L, about 4,000L, about 3,500 L, about 3,000 L, about 2,500 L, about 2,000 L, or about1,500 L (inclusive); between about 1,500 L and about 10,000 L, about9,500 L, about 9,000 L, about 8,500 L, about 8,000 L, about 7,500 L,about 7,000 L, about 6,500 L, about 6,000 L, about 5,500 L, about 5,000L, about 4,500 L, about 4,000 L, about 3,500 L, about 3,000 L, about2,500 L, or about 2,000 L (inclusive); between about 2,000 L and about10,000 L, about 9,500 L, about 9,000 L, about 8,500 L, about 8,000 L,about 7,500 L, about 7,000 L, about 6,500 L, about 6,000 L, about 5,500L, about 5,000 L, about 4,500 L, about 4,000 L, about 3,500 L, about3,000 L, or about 2,500 L (inclusive); between about 2,500 L and about10,000 L, about 9,500 L, about 9,000 L, about 8,500 L, about 8,000 L,about 7,500 L, about 7,000 L, about 6,500 L, about 6,000 L, about 5,500L, about 5,000 L, about 4,500 L, about 4,000 L, about 3,500 L, or about3,000 L (inclusive); between about 3,000 L and about 10,000 L, about9,500 L, about 9,000 L, about 8,500 L, about 8,000 L, about 7,500 L,about 7,000 L, about 6,500 L, about 6,000 L, about 5,500 L, about 5,000L, about 4,500 L, about 4,000 L, or about 3,500 L (inclusive); betweenabout 3,500 L and about 10,000 L, about 9,500 L, about 9,000 L, about8,500 L, about 8,000 L, about 7,500 L, about 7,000 L, about 6,500 L,about 6,000 L, about 5,500 L, about 5,000 L, about 4,500 L, or about4,000 L (inclusive); between about 4,000 L and about 10,000 L, about9,500 L, about 9,000 L, about 8,500 L, about 8,000 L, about 7,500 L,about 7,000 L, about 6,500 L, about 6,000 L, about 5,500 L, about 5,000L, or about 4,500 L (inclusive); between about 4,500 L and about 10,000L, about 9,500 L, about 9,000 L, about 8,500 L, about 8,000 L, about7,500 L, about 7,000 L, about 6,500 L, about 6,000 L, about 5,500 L, orabout 5,000 L (inclusive); between about 5,000 L and about 10,000 L,about 9,500 L, about 9,000 L, about 8,500 L, about 8,000 L, about 7,500L, about 7,000 L, about 6,500 L, about 6,000 L, or about 5,500 L(inclusive); between about 5,500 L and about 10,000 L, about 9,500 L,about 9,000 L, about 8,500 L, about 8,000 L, about 7,500 L, about 7,000L, about 6,500 L, or about 6,000 L (inclusive); between about 6,000 Land about 10,000 L, about 9,500 L, about 9,000 L, about 8,500 L, about8,000 L, about 7,500 L, about 7,000 L, or about 6,500 L (inclusive);between about 6,500 L and about 10,000 L, about 9,500 L, about 9,000 L,about 8,500 L, about 8,000 L, about 7,500 L, or about 7,000 L(inclusive); between about 7,000 L and about 10,000 L, about 9,500 L,about 9,000 L, about 8,500 L, about 8,000 L, or about 7,500 L(inclusive); between about 7,500 L and about 10,000 L, about 9,500 L,about 9,000 L, about 8,500 L, or about 8,000 L (inclusive); betweenabout 8,000 L and about 10,000 L, about 9,500 L, about 9,000 L, or about8,500 L (inclusive); between about 8,500 L and about 10,000 L, about9,500 L, or about 9,000 L (inclusive); between about 9,000 L and about10,000 L or about 9,500 L (inclusive); or between about 9,500 L andabout 10,000 L (inclusive).

Any reservoir(s) (e.g., break tank(s)) used (in any of the processesdescribed herein) to hold fluid before it enters into the first MCCS canhave a capacity that is, e.g., between 1 mL and about 100%, inclusive,e.g., between 1 mL and about 90%, about 80%, about 70%, about 60%, about50%, about 40%, about 30%, about 20%, about 10%, or about 5%, inclusive,of the loading volume of the first column of the first MCCS. Any of thereservoir(s) (e.g., break tanks(s)) used (in any of the processesdescribed herein) to hold fluid before it enters into the second MCCS(and after the fluid leaves the first MCCS) can have a capacity that is,e.g., between 1 mL and about 100%, inclusive, e.g., between 1 mL andabout 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about30%, about 20%, about 10%, or about 5%, inclusive, of the loading volumeof the first column of the second MCCS.

Various additional aspects of these processes are described in detailbelow and can be used in any combination in the processes providedherein without limitation. Exemplary aspects of the provided processesare described below; however, one skilled in the art will appreciatethat additional steps (e.g., the performance of additional unitoperations) can be added to the processes described herein and othermaterials can be used to perform any of the steps of the processesdescribed herein.

Liquid Culture Medium

Liquid culture medium that contains a recombinant therapeutic proteinthat is substantially free of cells can be derived from any source. Forexample, the liquid culture medium can be obtained from a recombinantcell culture (e.g., a recombinant bacterial, yeast, or mammalian cellculture). The liquid culture medium can be obtained from a fed-batchcell (e.g., mammalian cell) culture (e.g., a fed-batch bioreactorcontaining a culture of mammalian cells that secrete the recombinanttherapeutic protein) or a perfusion cell (e.g., mammalian cell) culture(e.g., a perfusion bioreactor containing a culture of mammalian cellsthat secrete the recombinant therapeutic protein). The liquid culturemedium can also be a clarified liquid culture medium from a culture ofbacterial or yeast cells that secrete the recombinant therapeuticprotein.

Liquid culture medium obtained from a recombinant cell culture can befiltered or clarified to obtain a liquid culture medium that issubstantially free of cells and/or viruses. Methods for filtering orclarifying a liquid culture medium in order to remove cells are known inthe art (e.g., 0.2-μm filtration and filtration using an AlternatingTangential Flow (ATF™) system).

Additional exemplary methods for filtering or clarifying a liquidculture medium include, e.g., immobilized cell separation (e.g., hollowfiber membranes, flat plate membranes, gel encapsulation, ceramicmatrices, and fluidized beds, e.g., as described in Kuhtreiber et al.,Springer Science & Business Media, 2013; Meuwly et al., Biotechnol. Adv.25(1):45-56, 2007; Jen et al., Biotechnol. Bioeng. 50(4):357-364, 1996;Piret et al., Biotechnol. Adv. 8(4):763-783, 1990; and Tyo et al.,Enzyme Microb. Technol. 9(9):514-520, 1987); density-based cellseparation (e.g., gravitational settling (e.g., gravitational settlingperformed using conical sedimentation columns (e.g., as described inKitano et al., Appl. Microbiol. Biotechnol. 24(4):282-286, 1986;Shintani et al., Cytotechnology 6(3):197-208, 1991), intra-reactorsettling zone (e.g., as described in Takazawa et al., Appl. Microbiol.Biotechnol. 32(3):280-284, 1989), a Dortmund settler (e.g., as describedin Hülscher et al., Biotechnol. Bioeng. 39(4):442-446, 1992), andinclined settlers (e.g., as described in Boycott et al., Nature 104:532,1920; Acrivos et al., J. Fluid Mechanics 92(3):435-457, 1979; Searles etal., Biotechnol. Prog. 10(2):198-206, 1994; Kohara et al., J. Chem. Eng.Japan 28(6):703-707, 1995; Shen et al., Biotechnol. Prog.27(5):1282-1296, 2011; Wang et al., Biotechnol. Prog. 26(5):1361-1366,2010; Pohlscheidt et al., Biotechnol. Prog. 29(1):222-229, 2013; Hechtet al., Biotechnol. Prog. 30(3):607-615, 2014)); centrifugation (e.g.,continuous centrifugation (e.g., as described in Hamamoto et al., J.Ferment. Bioeng. 67(3):190-194, 1989; Tokashiki et al., Cytotechnology3(3):239-244, 1990; and Takamatsu et al., Appl. Microbiol. Biotechnol.45(4):454-457, 1996), Centritech centrifugation (commercially availablefrom Pneumatic Scale Angelus and as described in, e.g., Johnson et al.,Biotechnol. Prog. 12(6):855-864, 1996; Kim et al., Biotechnol. Prog. 23(5): 1186-1197, 2007; Kim et al., Biotechnol. Prog. 24(1): 166-174,2008; Hecht et al., Biotechnol. Prog. 30 (3):607-615, 2014; Chotteau etal., Perfusion Processes, In: Al-Rubeai M (Ed.), Animal Cell Culture,Springer, Cham, Switzerland, 2015), and centrifuges that utilizecontinuous counterflow (e.g., as described in Pattasseril et al.,BioProcess Int. 11(3):38-47, 2013)); acoustic settling (e.g., using anacoustic settler, e.g., the acoustic settlers described in Kilburn etal., Biotechnol Bioeng. 34(4):559-562, 1989; Shirgaonkar et al.,Biotechnol. Adv. 22(6):433-444, 2004; Doblhoff-Dier et al., Biotechnol.Prog. 10(4):428-432, 1994; Gaida et al., Biotechnol. Prog. 12(1):73-76,1996; Bierau et al., J. Biotechnol. 62 (3):195-207, 1998; Crowley etal., BioProcess Int. 2(3):46-50, 2004; Gorenflo et al., Biotechnol.Prog. 19(1):30-36, 2003; Pui et al., Biotechnol. Prog. 11(2):146-152,1995; Gorenflo et al, Biotechnol. Bioeng. 80 (4):438-444, 2002; Dalm etal., Biotechnol. Bioeng. 88(5):547-557, 2004; Gorenflo et al.,Biotechnol. Prog. 19(1):30-36, 2005); and hydrocyclone separation (e.g.,using the hydroclone separators described in Elsayed et al., Int. J.Biotechnol. Wellness Indust. 2(4):153, 2013; Castilho et al., Continuousanimal cell perfusion processes: the first step toward integratedcontinuous biomanufacturing, in Subramanian G. (Ed.), ContinuousProcessing in Pharmaceutical Manufacturing, Wiley-VCH, Weinheim,Germany, 2014; Jockwer et al., The use of hydroclones for mammalian cellretention in perfusion bioreactors, in Animal Cell Technology: FromTarget to Market, Springer, pp. 301-306, 2001; Pinto et al.,Cytotechnology 56(1):57-67, 2008; Elsayed et al., Eng. Life Sci.6(4):347-354, 2006; Elsayed et al., BMC Proceedings 5(Supp. 8):p 65,2001; Castilho et al., Animal cell separation, in Castilho et al. (Ed.),Animal Cell Technology: From Biopharmaceuticals to Gene Therapy, Taylor& Francis, New York, N.Y., pp. 273-294, 2008).

Additional exemplary methods for filtering or clarifying a liquidculture medium include, e.g., size-based cell separation, e.g.,performed using spin filtration (e.g., spin filtration as described inHimmelfarb et al., Science 164(3879):555-557, 1969; Tolbert et al., InVitro 17(10):885-890, 1981; Reuveny et al., J. Immunol. Methods86(1):61-69, 1986; Avgerinos et al., Nat. Biotechnol. 8 (1):54-58, 1990;Kim et al., Biotechnol. Prog. 24(1):166-174, 2008; Bierau et al., J.Biotechnol. 62 (3):195-207, 1998; Castilho et al., Biotechnol. Prog.18(4):776-781, 2002; Deo et al., Biotechnol. Prog. 12(1):57-64, 1996;Emery et al., Appl. Microbiol. Biotechnol. 43(6):1028-1033, 1995; Hechtet al., Biotechnol. Prog. 30(3):607-615, 2014; Vallez-Chetreaunu et al.,J. Biotechnol. 130(3):265-273, 2007; Yabannavar et al., Biotechnol.Bioeng. 40 (8):925-933, 1992; Yabannavar et al., Biotechnol. Bioeng. 40(8):925-933, 1994; Deo et al., Biotechnol. Prog. 12 (1):57-64 1996; andCastilho et al., Biotechnol. Prog. 18(4):776-781, 2002); hollow-fiberfiltration (e.g., placement of hollow-fiber cartridges placed directlywithin a bioreactor (e.g., as described in Seamans et al., J. Ferment.Bioeng. 70(4):241-245, 1990, and Kyung et al., Cytotechnology14(3):183-190, 1994), tangential-flow-filtration (TFF) (e.g., asdescribed in Brennan et al., Biotechnol. Tech. 1(3):169-174, 1987; Velezet al., Biotechnol. Bioeng. 33(7):938-940, 1989; de la Broise et al.,Biotechnol. Bioeng. 38(7):781-787, 1991; Greenfield et al., BioprocessEng. 6(5):213-219, 1991; Kawahara et al., Cytotechnology 14(1):61-66,1994; Karst et al., Biochem. Eng. J 110:17-26, 2016; Martin et al., BMCProceedings 9:p 1, 2015; Clincke et al., Biotechnol. Prog. 29(3):754-767, 2013); alternating tangential flow (ATF) filtration (e.g.,as described in Kelly et al., Biotechnol. Prog. 30 (6):1291-1300, 2014;Karst et al., Biochem. Eng. J. 110:17-26, 2016; Kim et al.,Cytotechnology pp. 1-10, 2015; Xu et al., J. Biotechnol. 231:149-159,2016; Warikoo et al., Biotechnol. Bioeng. 109(12):3018-3029, 2012;Clincke et al., Biotechnol. Prog. 29(3):754-767, 2013; Wright et al.,Bioprocess International, Volume 13, Mar. 10, 2015; Yang et al.,Biotechnol. Prog. 30(3):616-625, 2014; and Padawer et al., Biotechnol.Prog. 29(3):829-832, 2013); use of a disposable floating filter (e.g.,Tao et al., Biotechnol. Prog. 27(3):824-829, 2011); vortex flowfiltration (VFF) (e.g., as described in Mercille et al., Biotechnol.Bioeng. 43 (9):833-846, 1994; Mercille et al., Biotechnol. Bioeng. 67(4):435-450, 2000; and Roth et al., Pharmaceutical Technology 21(10),1997); and continuous depth filtration (as described in Vogel et al.,Biotechnol. Bioeng. 109 (12):3049-3058, 2012, and Klutz et al., ChemicalEngineering Processing: Process Intensification 102:88-101, 2015).

Recombinant cells can also be removed from liquid culture medium usingcentrifugation and removing the supernatant that is liquid culturemedium that is substantially free of cells, or by allowing the cells tosettle to the gravitational bottom of a container (e.g., bioreactor)containing the liquid culture medium, and removing the liquid culturemedium (the liquid culture medium that is substantially free of cells)that is distant from the settled recombinant cells.

The liquid culture medium can be obtained from a culture of recombinantcells (e.g., recombinant bacteria, yeast, or mammalian cells) producingany of the recombinant therapeutic proteins described herein. Someexamples of any of the processes described herein can further include astep of culturing recombinant cells (e.g., recombinant bacteria, yeast,or mammalian cells) that produce the recombinant therapeutic protein.

The liquid culture medium can be any of the types of liquid culturemedium described herein or known in the art. For example, the liquidculture medium can be selected from the group of: animal-derivedcomponent free liquid culture medium, serum-free liquid culture medium,serum-containing liquid culture medium, chemically-defined liquidculture medium, and protein-free liquid culture medium. In any of theprocesses described herein, a liquid culture medium obtained from aculture can be diluted by addition of a second fluid (e.g., a buffer)before it is fed into the first MCCS (e.g., first PCCS).

The liquid culture medium containing a recombinant therapeutic proteinthat is substantially free of cells can be stored (e.g., at atemperature below about 15° C. (e.g., below about 10° C., below about 4°C., below about 0° C., below about −20° C., below about −50° C., belowabout −70 C.°, or below about −80° C.) for at least 1 day (e.g., atleast about 2 days, at least about 5 days, at least about 10 days, atleast about 15 days, at least about 20 days, or at least about 30 days)prior to feeding the liquid culture medium into the first MCCS (e.g.,first PCCS). Alternatively, in some examples the liquid culture mediumis fed into the first MCCS (e.g., first PCCS) directly from a bioreactor(e.g., fed into the first MCCS (e.g., first PCCS) directly from thebioreactor after a filtering or clarification step).

Recombinant Therapeutic Proteins

Non-limiting examples of recombinant therapeutic proteins that can beproduced by the methods provided herein include immunoglobulins(including light and heavy chain immunoglobulins, antibodies, orantibody fragments (e.g., any of the antibody fragments describedherein), enzymes (e.g., a galactosidase (e.g., an alpha-galactosidase),Myozyme, or Cerezyme), proteins (e.g., human erythropoietin, tumornecrosis factor (TNF), or an interferon alpha or beta), or immunogenicor antigenic proteins or protein fragments (e.g., proteins for use in avaccine). The recombinant therapeutic protein can be an engineeredantigen-binding polypeptide that contains at least one multifunctionalrecombinant protein scaffold (see, e.g., the recombinant antigen-bindingproteins described in Gebauer et al., Current Opin. Chem. Biol.13:245-255, 2009; and U.S. Patent Application Publication No.2012/0164066 (herein incorporated by reference in its entirety)).Non-limiting examples of recombinant therapeutic proteins that areantibodies include: panitumumab, omalizumab, abagovomab, abciximab,actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab,alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, amatuximab,anatumomab, anrukinzumab, apolizumab, arcitumomab, atinumab,tocilizumab, basilizimab, bectumomab, belimumab, bevacizumab,besilesomab, bezlotoxumab, biciromab, canakinumab, certolizumab,cetuximab, cixutumumab, daclizumab, denosumab, densumab, eculizumab,edrecolomab, efalizumab, efungumab, epratuzumab, ertumaxomab,etaracizumab, figitumumab, golimumab, ibritumomab tiuxetan, igovomab,imgatuzumab, infliximab, inolimomab, inotuzumab, labetuzumab,lebrikizumab, moxetumomab, natalizumab, obinutuzumab, oregovomab,palivizumab, panitumumab, pertuzumab, ranibizumab, rituximab,tocilizumab, tositumomab, tralokinumab, tucotuzumab, trastuzumab,veltuzumab, zalutumumab, and zatuximab. Additional examples ofrecombinant therapeutic antibodies that can be produced by the methodsdescribed herein are known in the art. Additional non-limiting examplesof recombinant therapeutic proteins that can be produced by the presentmethods include: alglucosidase alfa, laronidase, abatacept, galsulfase,lutropin alfa, antihemophilic factor, agalsidase beta, interferonbeta-la, darbepoetin alfa, tenecteplase, etanercept, coagulation factorIX, follicle stimulating hormone, interferon beta-la, imiglucerase,dornase alfa, epoetin alfa, insulin or insulin analogs, mecasermin,factov VIII, factor VIIa, anti-thrombin III, protein C, human albumin,erythropoietin, granulocute colony stimulating factor, granulocytemacrophage colony stimulating factor, interleukin-11, laronidase,idursuphase, galsulphase, α-1-proteinase inhibitor, lactase, adenosinedeaminase, tissue plasminogen activator, thyrotropin alpha (e.g.,Thyrogen®) and alteplase. Additional examples of recombinant proteinsthat can be produced by the present methods include acid α-glucosidase,alglucosidase alpha (e.g., Myozyme® and Lumizyme®), α-L-iduronidase(e.g., Aldurazyme®), iduronate sulfatase, heparan N-sulfatase,galactose-6-sulfatase, acid β-galactosidase, β-glucoronidase,N-acetylglucosamine-1-phosphotransferase, α-N-acetylgalactosaminidase,acid lipase, lysosomal acid ceramidase, acid sphingomyelinase,β-glucosidase (e.g., Cerezyme® and Ceredase®), galactosylceramidase,α-galactosidase-A (e.g., Fabrazyme®), acid β-galactosidase,β-galactosidase, neuraminidase, hexosaminidase A, and hexosaminidase B.

A secreted, soluble recombinant therapeutic protein can be recoveredfrom the liquid culture medium (e.g., a first and/or second liquidculture medium) by removing or otherwise physically separating theliquid culture medium from the cells (e.g., mammalian cells). A varietyof different methods for removing liquid culture medium from cells(e.g., mammalian cells) are known in the art, including, for example,centrifugation, filtration, pipetting, and/or aspiration. The secretedrecombinant therapeutic protein can then be recovered and furtherpurified from the liquid culture medium using a variety of biochemicaltechniques including various types of chromatography (e.g., affinitychromatography, molecular sieve chromatography, cation exchangechromatography, or anion exchange chromatography) and/or filtration(e.g., molecular weight cut-off filtration).

Multi-Column Chromatography Systems

The processes described herein include the use of two or more (e.g.,two, three, four, five, or six) multi-column chromatography systems(MCCSs). A MCCS can include two or more chromatography columns, two ormore chromatographic membranes, or a combination of at least onechromatography column and at least one chromatographic membrane. Innon-limiting examples, a MCCS (e.g., the first and/or second MCCSdescribed in any of the processes herein) can include fourchromatographic columns, three chromatographic columns and achromatographic membrane, three chromatographic columns, twochromatographic columns, two chromatographic membranes, and twochromatographic columns and one chromatographic membrane. Additionalexamples of combinations of chromatography columns and/orchromatographic membranes can be envisioned for use in an MCCS (e.g., afirst and/or second MCCS) by one skilled in the art without limitation.The individual chromatography columns and/or chromatographic membranespresent in a MCCS can be identical (e.g., have the same shape, volume,resin, capture mechanism, and unit operation), or can be different(e.g., have one or more of a different shape, volume, resin, capturemechanism, and unit operation). The individual chromatography column(s)and/or chromatographic membrane(s) present in a MCCS (e.g., the firstand/or second MCCS) can perform the same unit operation (e.g., the unitoperation of capturing, purifying, or polishing) or different unitoperations (e.g., different unit operations selected from, e.g., thegroup of capturing, purifying, polishing, inactivating viruses,adjusting the ionic concentration and/or pH of a fluid containing therecombinant therapeutic protein, and filtering).

The one or more chromatography column(s) that can be present in an MCCS(e.g., present in the first and/or second MCCS) can have a resin volumeof, e.g., at least about 2 mL, at least about 5 mL, at least about 10mL, at least about 15 mL, at least about 20 mL, at least about 25 mL, atleast about 30 mL, at least about 35 mL, at least about 40 mL, at leastabout 45 mL, at least about 50 mL, at least about 55 mL, at least about60 mL, at least about 65 mL, at least about 70 mL, at least about 75 mL,at least about 80 mL, at least about 85 mL, at least about 90 mL, atleast about 95 mL, at least about 100 mL, at least about 110 mL, atleast about 120 mL, at least about 130 mL, at least about 140 mL, atleast about 150 mL, at least about 160 mL, at least about 170 mL, atleast about 180 mL, at least about 190 mL, at least about 200 mL, atleast about 250 mL, at least about 300 mL, at least about 350 mL, atleast about 400 mL, at least about 450 mL, at least about 500 mL, atleast about 550 mL, at least about 600 mL, at least about 650 mL, atleast about 700 mL, at least about 750 mL, at least about 800 mL, atleast about 850 mL, at least about 900 mL, at least about 950 mL, atleast about 1 L, at least about 10 L, at least about 20 L, at leastabout 30 L, at least about 40 L, at least about 50 L, at least about 60L, at least about 70 L, at least about 80 L, at least about 90 L, atleast about 100 L, at least about 110 L, at least about 120 L, at leastabout 130 L, at least about 140 L, at least about 150 L, at least about160 L, at least about 170 L, at least about 180 L, at least about 190 L,or at least about 200 L.

The one or more chromatography column(s) that can be present in an MCCS(e.g., present in the first and/or second MCCS) can have a resin volumeof between about 2 mL and about 200 L, about 180 L, about 160 L, about140 L, about 120 L, about 100 L, about 80 L, about 60 L, about 40 L,about 20 L, about 10 L, about 5 L, about 1 L, about 900 mL, about 800mL, about 700 mL, about 600 mL, about 500 mL, about 400 mL, about 300mL, about 200 mL, about 100 mL, about 90 mL, about 80 mL, about 70 mL,about 60 mL, about 50 mL, about 45 mL, about 40 mL, about 35 mL, about30 mL, about 25 mL, about 20 mL, about 15 mL, about 10 mL, or about 5mL; between about 5 mL and about 200 L, about 180 L, about 160 L, about140 L, about 120 L, about 100 L, about 80 L, about 60 L, about 40 L,about 20 L, about 10 L, about 5 L, about 1 L, about 900 mL, about 800mL, about 700 mL, about 600 mL, about 500 mL, about 400 mL, about 300mL, about 200 mL, about 100 mL, about 90 mL, about 80 mL, about 70 mL,about 60 mL, about 50 mL, about 45 mL, about 40 mL, about 35 mL, about30 mL, about 25 mL, about 20 mL, about 15 mL, or about 10 mL; betweenabout 10 mL and about 200 L, about 180 L, about 160 L, about 140 L,about 120 L, about 100 L, about 80 L, about 60 L, about 40 L, about 20L, about 10 L, about 5 L, about 1 L, about 900 mL, about 800 mL, about700 mL, about 600 mL, about 500 mL, about 400 mL, about 300 mL, about200 mL, about 100 mL, about 90 mL, about 80 mL, about 70 mL, about 60mL, about 50 mL, about 45 mL, about 40 mL, about 35 mL, about 30 mL,about 25 mL, about 20 mL, or about 15 mL; between about 15 mL and about200 L, about 180 L, about 160 L, about 140 L, about 120 L, about 100 L,about 80 L, about 60 L, about 40 L, about 20 L, about 10 L, about 5 L,about 1 L, about 900 mL, about 800 mL, about 700 mL, about 600 mL, about500 mL, about 400 mL, about 300 mL, about 200 mL, about 100 mL, about 90mL, about 80 mL, about 70 mL, about 60 mL, about 50 mL, about 45 mL,about 40 mL, about 35 mL, about 30 mL, about 25 mL, or about 20 mL;between about 20 mL and about 200 L, about 180 L, about 160 L, about 140L, about 120 L, about 100 L, about 80 L, about 60 L, about 40 L, about20 L, about 10 L, about 5 L, about 1 L, about 900 mL, about 800 mL,about 700 mL, about 600 mL, about 500 mL, about 400 mL, about 300 mL,about 200 mL, about 100 mL, about 90 mL, about 80 mL, about 70 mL, about60 mL, about 50 mL, about 45 mL, about 40 mL, about 35 mL, about 30 mL,or about 25 mL; between about 25 mL and about 200 L, about 180 L, about160 L, about 140 L, about 120 L, about 100 L, about 80 L, about 60 L,about 40 L, about 20 L, about 10 L, about 5 L, about 1 L, about 900 mL,about 800 mL, about 700 mL, about 600 mL, about 500 mL, about 400 mL,about 300 mL, about 200 mL, about 100 mL, about 90 mL, about 80 mL,about 70 mL, about 60 mL, about 50 mL, about 45 mL, about 40 mL, about35 mL, or about 30 mL; between about 30 mL and about 200 L, about 180 L,about 160 L, about 140 L, about 120 L, about 100 L, about 80 L, about 60L, about 40 L, about 20 L, about 10 L, about 5 L, about 1 L, about 900mL, about 800 mL, about 700 mL, about 600 mL, about 500 mL, about 400mL, about 300 mL, about 200 mL, about 100 mL, about 90 mL, about 80 mL,about 70 mL, about 60 mL, about 50 mL, about 45 mL, about 40 mL, orabout 35 mL; between about 35 mL and about 200 L, about 180 L, about 160L, about 140 L, about 120 L, about 100 L, about 80 L, about 60 L, about40 L, about 20 L, about 10 L, about 5 L, about 1 L, about 900 mL, about800 mL, about 700 mL, about 600 mL, about 500 mL, about 400 mL, about300 mL, about 200 mL, about 100 mL, about 90 mL, about 80 mL, about 70mL, about 60 mL, about 50 mL, about 45 mL, or about 40 mL; between about40 mL and about 200 L, about 180 L, about 160 L, about 140 L, about 120L, about 100 L, about 80 L, about 60 L, about 40 L, about 20 L, about 10L, about 5 L, about 1 L, about 900 mL, about 800 mL, about 700 mL, about600 mL, about 500 mL, about 400 mL, about 300 mL, about 200 mL, about100 mL, about 90 mL, about 80 mL, about 70 mL, about 60 mL, about 50 mL,or about 45 mL; between about 45 mL and about 200 L, about 180 L, about160 L, about 140 L, about 120 L, about 100 L, about 80 L, about 60 L,about 40 L, about 20 L, about 10 L, about 5 L, about 1 L, about 900 mL,about 800 mL, about 700 mL, about 600 mL, about 500 mL, about 400 mL,about 300 mL, about 200 mL, about 100 mL, about 90 mL, about 80 mL,about 70 mL, about 60 mL, or about 50 mL; between about 50 mL and about200 L, about 180 L, about 160 L, about 140 L, about 120 L, about 100 L,about 80 L, about 60 L, about 40 L, about 20 L, about 10 L, about 5 L,about 1 L, about 900 mL, about 800 mL, about 700 mL, about 600 mL, about500 mL, about 400 mL, about 300 mL, about 200 mL, about 100 mL, about 90mL, about 80 mL, about 70 mL, or about 60 mL; between about 60 mL andabout 200 L, about 180 L, about 160 L, about 140 L, about 120 L, about100 L, about 80 L, about 60 L, about 40 L, about 20 L, about 10 L, about5 L, about 1 L, about 900 mL, about 800 mL, about 700 mL, about 600 mL,about 500 mL, about 400 mL, about 300 mL, about 200 mL, about 100 mL,about 90 mL, about 80 mL, or about 70 mL; between about 70 mL and about200 L, about 180 L, about 160 L, about 140 L, about 120 L, about 100 L,about 80 L, about 60 L, about 40 L, about 20 L, about 10 L, about 5 L,about 1 L, about 900 mL, about 800 mL, about 700 mL, about 600 mL, about500 mL, about 400 mL, about 300 mL, about 200 mL, about 100 mL, about 90mL, or about 80 mL; between about 80 mL and about 200 L, about 180 L,about 160 L, about 140 L, about 120 L, about 100 L, about 80 L, about 60L, about 40 L, about 20 L, about 10 L, about 5 L, about 1 L, about 900mL, about 800 mL, about 700 mL, about 600 mL, about 500 mL, about 400mL, about 300 mL, about 200 mL, about 100 mL, or about 90 mL; betweenabout 90 mL and about 200 L, about 180 L, about 160 L, about 140 L,about 120 L, about 100 L, about 80 L, about 60 L, about 40 L, about 20L, about 10 L, about 5 L, about 1 L, about 900 mL, about 800 mL, about700 mL, about 600 mL, about 500 mL, about 400 mL, about 300 mL, about200 mL, or about 100 mL; between about 100 mL and about 200 L, about 180L, about 160 L, about 140 L, about 120 L, about 100 L, about 80 L, about60 L, about 40 L, about 20 L, about 10 L, about 5 L, about 1 L, about900 mL, about 800 mL, about 700 mL, about 600 mL, about 500 mL, about400 mL, about 300 mL, or about 200 mL; between about 200 mL and about200 L, about 180 L, about 160 L, about 140 L, about 120 L, about 100 L,about 80 L, about 60 L, about 40 L, about 20 L, about 10 L, about 5 L,about 1 L, about 900 mL, about 800 mL, about 700 mL, about 600 mL, about500 mL, about 400 mL, or about 300 mL; about 300 mL and about 200 L,about 180 L, about 160 L, about 140 L, about 120 L, about 100 L, about80 L, about 60 L, about 40 L, about 20 L, about 10 L, about 5 L, about 1L, about 900 mL, about 800 mL, about 700 mL, about 600 mL, about 500 mL,or about 400 mL; between about 400 mL and about 200 L, about 180 L,about 160 L, about 140 L, about 120 L, about 100 L, about 80 L, about 60L, about 40 L, about 20 L, about 10 L, about 5 L, about 1 L, about 900mL, about 800 mL, about 700 mL, about 600 mL, or about 500 mL; betweenabout 500 mL and about 200 L, about 180 L, about 160 L, about 140 L,about 120 L, about 100 L, about 80 L, about 60 L, about 40 L, about 20L, about 10 L, about 5 L, about 1 L, about 900 mL, about 800 mL, about700 mL, or about 600 mL; between about 600 mL and about 200 L, about 180L, about 160 L, about 140 L, about 120 L, about 100 L, about 80 L, about60 L, about 40 L, about 20 L, about 10 L, about 5 L, about 1 L, about900 mL, about 800 mL, or about 700 mL; between about 700 mL and about200 L, about 180 L, about 160 L, about 140 L, about 120 L, about 100 L,about 80 L, about 60 L, about 40 L, about 20 L, about 10 L, about 5 L,about 1 L, about 900 mL, or about 800 mL; between about 800 mL and about200 L, about 180 L, about 160 L, about 140 L, about 120 L, about 100 L,about 80 L, about 60 L, about 40 L, about 20 L, about 10 L, about 5 L,about 1 L, or about 900 mL; between about 900 mL and about 200 L, about180 L, about 160 L, about 140 L, about 120 L, about 100 L, about 80 L,about 60 L, about 40 L, about 20 L, about 10 L, about 5 L, or about 1 L;between about 1 L and about 200 L, about 180 L, about 160 L, about 140L, about 120 L, about 100 L, about 80 L, about 60 L, about 40 L, about20 L, about 10 L, or about 5 L; between about 5 L and about 200 L, about180 L, about 160 L, about 140 L, about 120 L, about 100 L, about 80 L,about 60 L, about 40 L, about 20 L, or about 10 L; between 10 L andabout 200 L, about 180 L, about 160 L, about 140 L, about 120 L, about100 L, about 80 L, about 60 L, about 40 L, or about 20 L; between 20 Land about 200 L, about 180 L, about 160 L, about 140 L, about 120 L,about 100 L, about 80 L, about 60 L, or about 40 L; between about 40 Land about 200 L, about 180 L, about 160 L, about 140 L, about 120 L,about 100 L, about 80 L, or about 60 L; between about 60 L and about 200L, about 180 L, about 160 L, about 140 L, about 120 L, about 100 L, orabout 80 L; between about 80 L and about 200 L, about 180 L, about 160L, about 140 L, about 120 L, or about 100 L; between about 100 L andabout 200 L, about 180 L, about 160 L, about 140 L, or about 120 L;between about 120 L and about 200 L, about 180 L, about 160 L, or about140 L; between about 140 L and about 200 L, about 180 L, or about 160 L;between about 160 L and about 200 L or about 180 L; or about 180 L toabout 200 L.

The one or more chromatography column(s) in an MCCS (e.g., the firstand/or second MCCS) used in any of the processes described herein canhave the substantially the same resin volume or can have different resinvolumes. The flow rate used for the one or more chromatography column(s)in an MCCS (e.g., the first and/or second MCCS) can be, e.g., betweenabout 0.2 mL/minute to about 100 L/minute, about 80 L/minute, about 60L/minute, about 40 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 900 mL/minute, about 800 mL/minute,about 700 mL/minute, about 600 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 90 mL/minute, about 80 mL/minute,about 70 mL/minute, about 60 mL/minute, about 50 mL/minute, about 40mL/minute, about 30 mL/minute, about 25 mL/minute, about 20 mL/minute,about 15 mL/minute, about 10 mL/minute, about 5 mL/minute, about 2mL/minute, or about 1 mL/minute; between about 1 mL/minute to about 100L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute, about20 L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute,about 900 mL/minute, about 800 mL/minute, about 700 mL/minute, about 600mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 90 mL/minute, about 80 mL/minute, about 70 mL/minute,about 60 mL/minute, about 50 mL/minute, about 40 mL/minute, about 30mL/minute, about 25 mL/minute, about 20 mL/minute, about 15 mL/minute,about 10 mL/minute, about 5 mL/minute, or about 2 mL/minute; betweenabout 2 mL/minute to about 100 L/minute, about 80 L/minute, about 60L/minute, about 40 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 900 mL/minute, about 800 mL/minute,about 700 mL/minute, about 600 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 90 mL/minute, about 80 mL/minute,about 70 mL/minute, about 60 mL/minute, about 50 mL/minute, about 40mL/minute, about 30 mL/minute, about 25 mL/minute, about 20 mL/minute,about 15 mL/minute, about 10 mL/minute, or about 5 mL/minute; betweenabout 5 mL/minute to about 100 L/minute, about 80 L/minute, about 60L/minute, about 40 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 900 mL/minute, about 800 mL/minute,about 700 mL/minute, about 600 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 90 mL/minute, about 80 mL/minute,about 70 mL/minute, about 60 mL/minute, about 50 mL/minute, about 40mL/minute, about 30 mL/minute, about 25 mL/minute, about 20 mL/minute,about 15 mL/minute, or about 10 mL/minute; between about 10 mL/minute toabout 100 L/minute, about 80 L/minute, about 60 L/minute, about 40L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 900 mL/minute, about 800 mL/minute, about 700mL/minute, about 600 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 90 mL/minute, about 80 mL/minute,about 70 mL/minute, about 60 mL/minute, about 50 mL/minute, about 40mL/minute, about 30 mL/minute, about 25 mL/minute, about 20 mL/minute,or about 15 mL/minute; between about 15 mL/minute to about 100 L/minute,about 80 L/minute, about 60 L/minute, about 40 L/minute, about 20L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about900 mL/minute, about 800 mL/minute, about 700 mL/minute, about 600mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 90 mL/minute, about 80 mL/minute, about 70 mL/minute,about 60 mL/minute, about 50 mL/minute, about 40 mL/minute, about 30mL/minute, about 25 mL/minute, or about 20 mL/minute; between about 20mL/minute to about 100 L/minute, about 80 L/minute, about 60 L/minute,about 40 L/minute, about 20 L/minute, about 10 L/minute, about 5L/minute, about 1 L/minute, about 900 mL/minute, about 800 mL/minute,about 700 mL/minute, about 600 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 90 mL/minute, about 80 mL/minute,about 70 mL/minute, about 60 mL/minute, about 50 mL/minute, about 40mL/minute, about 30 mL/minute, or about 25 mL/minute; between about 25mL/minute to about 100 L/minute, about 80 L/minute, about 60 L/minute,about 40 L/minute, about 20 L/minute, about 10 L/minute, about 5L/minute, about 1 L/minute, about 900 mL/minute, about 800 mL/minute,about 700 mL/minute, about 600 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 90 mL/minute, about 80 mL/minute,about 70 mL/minute, about 60 mL/minute, about 50 mL/minute, about 40mL/minute, or about 30 mL/minute; between about 30 mL/minute to about100 L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 900 mL/minute, about 800 mL/minute, about 700 mL/minute,about 600 mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 90 mL/minute, about 80 mL/minute, about 70 mL/minute,about 60 mL/minute, about 50 mL/minute, or about 40 mL/minute; betweenabout 40 mL/minute to about 100 L/minute, about 80 L/minute, about 60L/minute, about 40 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 900 mL/minute, about 800 mL/minute,about 700 mL/minute, about 600 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 90 mL/minute, about 80 mL/minute,about 70 mL/minute, about 60 mL/minute, or about 50 mL/minute; betweenabout 50 mL/minute to about 100 L/minute, about 80 L/minute, about 60L/minute, about 40 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 900 mL/minute, about 800 mL/minute,about 700 mL/minute, about 600 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 90 mL/minute, about 80 mL/minute,about 70 mL/minute, or about 60 mL/minute; between about 60 mL/minute toabout 100 L/minute, about 80 L/minute, about 60 L/minute, about 40L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 900 mL/minute, about 800 mL/minute, about 700mL/minute, about 600 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 90 mL/minute, about 80 mL/minute,or about 70 mL/minute; between about 70 mL/minute to about 100 L/minute,about 80 L/minute, about 60 L/minute, about 40 L/minute, about 20L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about900 mL/minute, about 800 mL/minute, about 700 mL/minute, about 600mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 90 mL/minute, or about 80 mL/minute; between about 80mL/minute to about 100 L/minute, about 80 L/minute, about 60 L/minute,about 40 L/minute, about 20 L/minute, about 10 L/minute, about 5L/minute, about 1 L/minute, about 900 mL/minute, about 800 mL/minute,about 700 mL/minute, about 600 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, or about 90 mL/minute; between about 90mL/minute to about 100 L/minute, about 80 L/minute, about 60 L/minute,about 40 L/minute, about 20 L/minute, about 10 L/minute, about 5L/minute, about 1 L/minute, about 900 mL/minute, about 800 mL/minute,about 700 mL/minute, about 600 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, or about 100 mL/minute; between about 100 mL/minute to about100 L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 900 mL/minute, about 800 mL/minute, about 700 mL/minute,about 600 mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, or about 150 mL/minute; between about150 mL/minute to about 100 L/minute, about 80 L/minute, about 60L/minute, about 40 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 900 mL/minute, about 800 mL/minute,about 700 mL/minute, about 600 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, or about 200 mL/minute; between about200 mL/minute to about 100 L/minute, about 80 L/minute, about 60L/minute, about 40 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 900 mL/minute, about 800 mL/minute,about 700 mL/minute, about 600 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, or about 250 mL/minute; between about 250 mL/minute to about100 L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 900 mL/minute, about 800 mL/minute, about 700 mL/minute,about 600 mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, or about 300 mL/minute; between about300 mL/minute to about 100 L/minute, about 80 L/minute, about 60L/minute, about 40 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 900 mL/minute, about 800 mL/minute,about 700 mL/minute, about 600 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, or about 350 mL/minute; between about350 mL/minute to about 100 L/minute, about 80 L/minute, about 60L/minute, about 40 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 900 mL/minute, about 800 mL/minute,about 700 mL/minute, about 600 mL/minute, about 500 mL/minute, about 450mL/minute, or about 400 mL/minute; between about 400 mL/minute to about100 L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, about 900 mL/minute, about 800 mL/minute, about 700 mL/minute,about 600 mL/minute, about 500 mL/minute, or about 450 mL/minute;between about 450 mL/minute to about 100 L/minute, about 80 L/minute,about 60 L/minute, about 40 L/minute, about 20 L/minute, about 10L/minute, about 5 L/minute, about 1 L/minute, about 900 mL/minute, about800 mL/minute, about 700 mL/minute, about 600 mL/minute, or about 500mL/minute; between about 500 mL/minute to about 100 L/minute, about 80L/minute, about 60 L/minute, about 40 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 900 mL/minute,about 800 mL/minute, about 700 mL/minute, or about 600 mL/minute;between about 600 mL/minute to about 100 L/minute, about 80 L/minute,about 60 L/minute, about 40 L/minute, about 20 L/minute, about 10L/minute, about 5 L/minute, about 1 L/minute, about 900 mL/minute, about800 mL/minute, or about 700 mL/minute; between about 700 mL/minute toabout 100 L/minute, about 80 L/minute, about 60 L/minute, about 40L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 900 mL/minute, or about 800 mL/minute; between about800 mL/minute to about 100 L/minute, about 80 L/minute, about 60L/minute, about 40 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, or about 900 mL/minute; between about 900mL/minute to about 100 L/minute, about 80 L/minute, about 60 L/minute,about 40 L/minute, about 20 L/minute, about 10 L/minute, about 5L/minute, or about 1 L/minute; between about 1 L/minute to about 100L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute, about20 L/minute, about 10 L/minute, or about 5 L/minute; about 5 L/minute toabout 100 L/minute, about 80 L/minute, about 60 L/minute, about 40L/minute, about 20 L/minute, or about 10 L/minute; between about 10L/minute to about 100 L/minute, about 80 L/minute, about 60 L/minute,about 40 L/minute, or about 20 L/minute; between about 20 L/minute toabout 100 L/minute, about 80 L/minute, about 60 L/minute, or about 40L/minute; between about 40 L/minute to about 100 L/minute, about 80L/minute, or about 60 L/minute; between about 60 L/minute to about 100L/minute or about 80 L/minute; or about 80 L/minute to about 100L/minute.

The one or more chromatography column (s) in an MCCS (e.g., the firstand/or second MCCS) can have substantially the same shape or can havesubstantially different shapes. For example, the one or morechromatography column(s) in an MCCS (e.g., in the first and/or secondMCCS) can have substantially the shape of a circular cylinder or canhave substantially the same shape of an oval cylinder.

The one or more chromatographic membrane(s) that can be present in anMCCS (e.g., present in the first and/or second MCCS) can have a bedvolume of, e.g., between about 1 mL and about 10 L, about 9.5 L, about 9L, about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6L, about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3L, about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950 mL, about900 mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL, about650 mL, about 600 mL, about 550 mL, about 500 mL, about 475 mL, about450 mL, about 425 mL, about 400 mL, about 375 mL, about 350 mL, about325 mL, about 300 mL, about 275 mL, about 250 mL, about 225 mL, about200 mL, about 175 mL, about 150 mL, about 125 mL, about 100 mL, about 95mL, about 90 mL, about 85 mL, about 80 mL, about 75 mL, about 70 mL,about 65 mL, about 60 mL, about 55 mL, about 50 mL, about 45 mL, about40 mL, about 35 mL, about 30 mL, about 25 mL, about 20 mL, about 15 mL,about 10 mL, or about 5 mL (inclusive); between about 2 mL and about 10L, about 9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L, about 7L, about 6.5 L, about 6 L, about 5.5 L, about 5 L, about 4.5 L, about 4L, about 3.5 L, about 3 L, about 2.5 L, about 2 L, about 1.5 L, about 1L, about 950 mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL,about 700 mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL,about 475 mL, about 450 mL, about 425 mL, about 400 mL, about 375 mL,about 350 mL, about 325 mL, about 300 mL, about 275 mL, about 250 mL,about 225 mL, about 200 mL, about 175 mL, about 150 mL, about 125 mL,about 100 mL, about 95 mL, about 90 mL, about 85 mL, about 80 mL, about75 mL, about 70 mL, about 65 mL, about 60 mL, about 55 mL, about 50 mL,about 45 mL, about 40 mL, about 35 mL, about 30 mL, about 25 mL, about20 mL, about 15 mL, about 10 mL, or about 5 mL (inclusive); betweenabout 5 mL and about 10 L, about 9.5 L, about 9 L, about 8.5 L, about 8L, about 7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5 L, about 5L, about 4.5 L, about 4 L, about 3.5 L, about 3 L, about 2.5 L, about 2L, about 1.5 L, about 1 L, about 950 mL, about 900 mL, about 850 mL,about 800 mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL,about 550 mL, about 500 mL, about 475 mL, about 450 mL, about 425 mL,about 400 mL, about 375 mL, about 350 mL, about 325 mL, about 300 mL,about 275 mL, about 250 mL, about 225 mL, about 200 mL, about 175 mL,about 150 mL, about 125 mL, about 100 mL, about 95 mL, about 90 mL,about 85 mL, about 80 mL, about 75 mL, about 70 mL, about 65 mL, about60 mL, about 55 mL, about 50 mL, about 45 mL, about 40 mL, about 35 mL,about 30 mL, about 25 mL, about 20 mL, about 15 mL, or about 10 mL(inclusive); between about 10 mL and about 10 L, about 9.5 L, about 9 L,about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6 L,about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3 L,about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950 mL, about 900mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL, about 650mL, about 600 mL, about 550 mL, about 500 mL, about 475 mL, about 450mL, about 425 mL, about 400 mL, about 375 mL, about 350 mL, about 325mL, about 300 mL, about 275 mL, about 250 mL, about 225 mL, about 200mL, about 175 mL, about 150 mL, about 125 mL, about 100 mL, about 95 mL,about 90 mL, about 85 mL, about 80 mL, about 75 mL, about 70 mL, about65 mL, about 60 mL, about 55 mL, about 50 mL, about 45 mL, about 40 mL,about 35 mL, about 30 mL, about 25 mL, about 20 mL, or about 15 mL(inclusive); between about 15 mL and about 10 L, about 9.5 L, about 9 L,about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6 L,about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3 L,about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950 mL, about 900mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL, about 650mL, about 600 mL, about 550 mL, about 500 mL, about 475 mL, about 450mL, about 425 mL, about 400 mL, about 375 mL, about 350 mL, about 325mL, about 300 mL, about 275 mL, about 250 mL, about 225 mL, about 200mL, about 175 mL, about 150 mL, about 125 mL, about 100 mL, about 95 mL,about 90 mL, about 85 mL, about 80 mL, about 75 mL, about 70 mL, about65 mL, about 60 mL, about 55 mL, about 50 mL, about 45 mL, about 40 mL,about 35 mL, about 30 mL, about 25 mL, or about 20 mL (inclusive);between about 20 mL and about 10 L, about 9.5 L, about 9 L, about 8.5 L,about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5 L,about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3 L, about 2.5 L,about 2 L, about 1.5 L, about 1 L, about 950 mL, about 900 mL, about 850mL, about 800 mL, about 750 mL, about 700 mL, about 650 mL, about 600mL, about 550 mL, about 500 mL, about 475 mL, about 450 mL, about 425mL, about 400 mL, about 375 mL, about 350 mL, about 325 mL, about 300mL, about 275 mL, about 250 mL, about 225 mL, about 200 mL, about 175mL, about 150 mL, about 125 mL, about 100 mL, about 95 mL, about 90 mL,about 85 mL, about 80 mL, about 75 mL, about 70 mL, about 65 mL, about60 mL, about 55 mL, about 50 mL, about 45 mL, about 40 mL, about 35 mL,about 30 mL, or about 25 mL (inclusive); between about 25 mL and about10 L, about 9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L, about7 L, about 6.5 L, about 6 L, about 5.5 L, about 5 L, about 4.5 L, about4 L, about 3.5 L, about 3 L, about 2.5 L, about 2 L, about 1.5 L, about1 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL, about 750mL, about 700 mL, about 650 mL, about 600 mL, about 550 mL, about 500mL, about 475 mL, about 450 mL, about 425 mL, about 400 mL, about 375mL, about 350 mL, about 325 mL, about 300 mL, about 275 mL, about 250mL, about 225 mL, about 200 mL, about 175 mL, about 150 mL, about 125mL, about 100 mL, about 95 mL, about 90 mL, about 85 mL, about 80 mL,about 75 mL, about 70 mL, about 65 mL, about 60 mL, about 55 mL, about50 mL, about 45 mL, about 40 mL, about 35 mL, or about 30 mL(inclusive); between about 30 mL and about 10 L, about 9.5 L, about 9 L,about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6 L,about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3 L,about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950 mL, about 900mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL, about 650mL, about 600 mL, about 550 mL, about 500 mL, about 475 mL, about 450mL, about 425 mL, about 400 mL, about 375 mL, about 350 mL, about 325mL, about 300 mL, about 275 mL, about 250 mL, about 225 mL, about 200mL, about 175 mL, about 150 mL, about 125 mL, about 100 mL, about 95 mL,about 90 mL, about 85 mL, about 80 mL, about 75 mL, about 70 mL, about65 mL, about 60 mL, about 55 mL, about 50 mL, about 45 mL, about 40 mL,or about 35 mL (inclusive); between about 35 mL and about 10 L, about9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L, about 7 L, about6.5 L, about 6 L, about 5.5 L, about 5 L, about 4.5 L, about 4 L, about3.5 L, about 3 L, about 2.5 L, about 2 L, about 1.5 L, about 1 L, about950 mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL, about700 mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL, about475 mL, about 450 mL, about 425 mL, about 400 mL, about 375 mL, about350 mL, about 325 mL, about 300 mL, about 275 mL, about 250 mL, about225 mL, about 200 mL, about 175 mL, about 150 mL, about 125 mL, about100 mL, about 95 mL, about 90 mL, about 85 mL, about 80 mL, about 75 mL,about 70 mL, about 65 mL, about 60 mL, about 55 mL, about 50 mL, about45 mL, or about 40 mL (inclusive); between about 40 mL and about 10 L,about 9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L, about 7 L,about 6.5 L, about 6 L, about 5.5 L, about 5 L, about 4.5 L, about 4 L,about 3.5 L, about 3 L, about 2.5 L, about 2 L, about 1.5 L, about 1 L,about 950 mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL,about 700 mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL,about 475 mL, about 450 mL, about 425 mL, about 400 mL, about 375 mL,about 350 mL, about 325 mL, about 300 mL, about 275 mL, about 250 mL,about 225 mL, about 200 mL, about 175 mL, about 150 mL, about 125 mL,about 100 mL, about 95 mL, about 90 mL, about 85 mL, about 80 mL, about75 mL, about 70 mL, about 65 mL, about 60 mL, about 55 mL, about 50 mL,or about 45 mL (inclusive); between about 45 mL and about 10 L, about9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L, about 7 L, about6.5 L, about 6 L, about 5.5 L, about 5 L, about 4.5 L, about 4 L, about3.5 L, about 3 L, about 2.5 L, about 2 L, about 1.5 L, about 1 L, about950 mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL, about700 mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL, about475 mL, about 450 mL, about 425 mL, about 400 mL, about 375 mL, about350 mL, about 325 mL, about 300 mL, about 275 mL, about 250 mL, about225 mL, about 200 mL, about 175 mL, about 150 mL, about 125 mL, about100 mL, about 95 mL, about 90 mL, about 85 mL, about 80 mL, about 75 mL,about 70 mL, about 65 mL, about 60 mL, about 55 mL, or about 50 mL(inclusive); between about 50 mL and about 10 L, about 9.5 L, about 9 L,about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6 L,about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3 L,about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950 mL, about 900mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL, about 650mL, about 600 mL, about 550 mL, about 500 mL, about 475 mL, about 450mL, about 425 mL, about 400 mL, about 375 mL, about 350 mL, about 325mL, about 300 mL, about 275 mL, about 250 mL, about 225 mL, about 200mL, about 175 mL, about 150 mL, about 125 mL, about 100 mL, about 95 mL,about 90 mL, about 85 mL, about 80 mL, about 75 mL, about 70 mL, about65 mL, about 60 mL, or about 55 mL (inclusive); between about 55 mL andabout 10 L, about 9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L,about 7 L, about 6.5 L, about 6 L, about 5.5 L, about 5 L, about 4.5 L,about 4 L, about 3.5 L, about 3 L, about 2.5 L, about 2 L, about 1.5 L,about 1 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL, about750 mL, about 700 mL, about 650 mL, about 600 mL, about 550 mL, about500 mL, about 475 mL, about 450 mL, about 425 mL, about 400 mL, about375 mL, about 350 mL, about 325 mL, about 300 mL, about 275 mL, about250 mL, about 225 mL, about 200 mL, about 175 mL, about 150 mL, about125 mL, about 100 mL, about 95 mL, about 90 mL, about 85 mL, about 80mL, about 75 mL, about 70 mL, about 65 mL, or about 60 mL (inclusive);between about 60 mL and about 10 L, about 9.5 L, about 9 L, about 8.5 L,about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5 L,about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3 L, about 2.5 L,about 2 L, about 1.5 L, about 1 L, about 950 mL, about 900 mL, about 850mL, about 800 mL, about 750 mL, about 700 mL, about 650 mL, about 600mL, about 550 mL, about 500 mL, about 475 mL, about 450 mL, about 425mL, about 400 mL, about 375 mL, about 350 mL, about 325 mL, about 300mL, about 275 mL, about 250 mL, about 225 mL, about 200 mL, about 175mL, about 150 mL, about 125 mL, about 100 mL, about 95 mL, about 90 mL,about 85 mL, about 80 mL, about 75 mL, about 70 mL, or about 65 mL(inclusive); between about 65 mL and about 10 L, about 9.5 L, about 9 L,about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6 L,about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3 L,about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950 mL, about 900mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL, about 650mL, about 600 mL, about 550 mL, about 500 mL, about 475 mL, about 450mL, about 425 mL, about 400 mL, about 375 mL, about 350 mL, about 325mL, about 300 mL, about 275 mL, about 250 mL, about 225 mL, about 200mL, about 175 mL, about 150 mL, about 125 mL, about 100 mL, about 95 mL,about 90 mL, about 85 mL, about 80 mL, about 75 mL, or about 70 mL(inclusive); between about 70 mL and about 10 L, about 9.5 L, about 9 L,about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6 L,about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3 L,about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950 mL, about 900mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL, about 650mL, about 600 mL, about 550 mL, about 500 mL, about 475 mL, about 450mL, about 425 mL, about 400 mL, about 375 mL, about 350 mL, about 325mL, about 300 mL, about 275 mL, about 250 mL, about 225 mL, about 200mL, about 175 mL, about 150 mL, about 125 mL, about 100 mL, about 95 mL,about 90 mL, about 85 mL, about 80 mL, or about 75 mL (inclusive);between about 75 mL and about 10 L, about 9.5 L, about 9 L, about 8.5 L,about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5 L,about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3 L, about 2.5 L,about 2 L, about 1.5 L, about 1 L, about 950 mL, about 900 mL, about 850mL, about 800 mL, about 750 mL, about 700 mL, about 650 mL, about 600mL, about 550 mL, about 500 mL, about 475 mL, about 450 mL, about 425mL, about 400 mL, about 375 mL, about 350 mL, about 325 mL, about 300mL, about 275 mL, about 250 mL, about 225 mL, about 200 mL, about 175mL, about 150 mL, about 125 mL, about 100 mL, about 95 mL, about 90 mL,about 85 mL, or about 80 mL (inclusive); between about 80 mL and about10 L, about 9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L, about7 L, about 6.5 L, about 6 L, about 5.5 L, about 5 L, about 4.5 L, about4 L, about 3.5 L, about 3 L, about 2.5 L, about 2 L, about 1.5 L, about1 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL, about 750mL, about 700 mL, about 650 mL, about 600 mL, about 550 mL, about 500mL, about 475 mL, about 450 mL, about 425 mL, about 400 mL, about 375mL, about 350 mL, about 325 mL, about 300 mL, about 275 mL, about 250mL, about 225 mL, about 200 mL, about 175 mL, about 150 mL, about 125mL, about 100 mL, about 95 mL, about 90 mL, or about 85 mL (inclusive);between about 85 mL and about 10 L, about 9.5 L, about 9 L, about 8.5 L,about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5 L,about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3 L, about 2.5 L,about 2 L, about 1.5 L, about 1 L, about 950 mL, about 900 mL, about 850mL, about 800 mL, about 750 mL, about 700 mL, about 650 mL, about 600mL, about 550 mL, about 500 mL, about 475 mL, about 450 mL, about 425mL, about 400 mL, about 375 mL, about 350 mL, about 325 mL, about 300mL, about 275 mL, about 250 mL, about 225 mL, about 200 mL, about 175mL, about 150 mL, about 125 mL, about 100 mL, about 95 mL, or about 90mL (inclusive); between about 90 mL and about 10 L, about 9.5 L, about 9L, about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6L, about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3L, about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950 mL, about900 mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL, about650 mL, about 600 mL, about 550 mL, about 500 mL, about 475 mL, about450 mL, about 425 mL, about 400 mL, about 375 mL, about 350 mL, about325 mL, about 300 mL, about 275 mL, about 250 mL, about 225 mL, about200 mL, about 175 mL, about 150 mL, about 125 mL, about 100 mL, or about95 mL (inclusive); between about 95 mL and about 10 L, about 9.5 L,about 9 L, about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L,about 6 L, about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L,about 3 L, about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950 mL,about 900 mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL,about 650 mL, about 600 mL, about 550 mL, about 500 mL, about 475 mL,about 450 mL, about 425 mL, about 400 mL, about 375 mL, about 350 mL,about 325 mL, about 300 mL, about 275 mL, about 250 mL, about 225 mL,about 200 mL, about 175 mL, about 150 mL, about 125 mL, or about 100 mL;between about 100 mL and about 10 L, about 9.5 L, about 9 L, about 8.5L, about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5L, about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3 L, about 2.5L, about 2 L, about 1.5 L, about 1 L, about 950 mL, about 900 mL, about850 mL, about 800 mL, about 750 mL, about 700 mL, about 650 mL, about600 mL, about 550 mL, about 500 mL, about 475 mL, about 450 mL, about425 mL, about 400 mL, about 375 mL, about 350 mL, about 325 mL, about300 mL, about 275 mL, about 250 mL, about 225 mL, about 200 mL, about175 mL, about 150 mL, or about 125 mL (inclusive); between about 125 mLand about 10 L, about 9.5 L, about 9 L, about 8.5 L, about 8 L, about7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5 L, about 5 L, about4.5 L, about 4 L, about 3.5 L, about 3 L, about 2.5 L, about 2 L, about1.5 L, about 1 L, about 950 mL, about 900 mL, about 850 mL, about 800mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL, about 550mL, about 500 mL, about 475 mL, about 450 mL, about 425 mL, about 400mL, about 375 mL, about 350 mL, about 325 mL, about 300 mL, about 275mL, about 250 mL, about 225 mL, about 200 mL, about 175 mL, or about 150mL (inclusive); between about 150 mL and about 10 L, about 9.5 L, about9 L, about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L, about6 L, about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L, about3 L, about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950 mL, about900 mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL, about650 mL, about 600 mL, about 550 mL, about 500 mL, about 475 mL, about450 mL, about 425 mL, about 400 mL, about 375 mL, about 350 mL, about325 mL, about 300 mL, about 275 mL, about 250 mL, about 225 mL, about200 mL, or about 175 mL (inclusive); between about 175 mL and about 10L, about 9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L, about 7L, about 6.5 L, about 6 L, about 5.5 L, about 5 L, about 4.5 L, about 4L, about 3.5 L, about 3 L, about 2.5 L, about 2 L, about 1.5 L, about 1L, about 950 mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL,about 700 mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL,about 475 mL, about 450 mL, about 425 mL, about 400 mL, about 375 mL,about 350 mL, about 325 mL, about 300 mL, about 275 mL, about 250 mL,about 225 mL, or about 200 mL (inclusive); between about 200 mL andabout 10 L, about 9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L,about 7 L, about 6.5 L, about 6 L, about 5.5 L, about 5 L, about 4.5 L,about 4 L, about 3.5 L, about 3 L, about 2.5 L, about 2 L, about 1.5 L,about 1 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL, about750 mL, about 700 mL, about 650 mL, about 600 mL, about 550 mL, about500 mL, about 475 mL, about 450 mL, about 425 mL, about 400 mL, about375 mL, about 350 mL, about 325 mL, about 300 mL, about 275 mL, about250 mL, or about 225 mL (inclusive); between about 225 mL and about 10L, about 9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L, about 7L, about 6.5 L, about 6 L, about 5.5 L, about 5 L, about 4.5 L, about 4L, about 3.5 L, about 3 L, about 2.5 L, about 2 L, about 1.5 L, about 1L, about 950 mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL,about 700 mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL,about 475 mL, about 450 mL, about 425 mL, about 400 mL, about 375 mL,about 350 mL, about 325 mL, about 300 mL, about 275 mL, or about 250 mL(inclusive); between about 250 mL and about 10 L, about 9.5 L, about 9L, about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6L, about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3L, about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950 mL, about900 mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL, about650 mL, about 600 mL, about 550 mL, about 500 mL, about 475 mL, about450 mL, about 425 mL, about 400 mL, about 375 mL, about 350 mL, about325 mL, about 300 mL, or about 275 mL (inclusive); between about 275 mLand about 10 L, about 9.5 L, about 9 L, about 8.5 L, about 8 L, about7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5 L, about 5 L, about4.5 L, about 4 L, about 3.5 L, about 3 L, about 2.5 L, about 2 L, about1.5 L, about 1 L, about 950 mL, about 900 mL, about 850 mL, about 800mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL, about 550mL, about 500 mL, about 475 mL, about 450 mL, about 425 mL, about 400mL, about 375 mL, about 350 mL, about 325 mL, or about 300 mL(inclusive); between about 300 mL and about 10 L, about 9.5 L, about 9L, about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6L, about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3L, about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950 mL, about900 mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL, about650 mL, about 600 mL, about 550 mL, about 500 mL, about 475 mL, about450 mL, about 425 mL, about 400 mL, about 375 mL, about 350 mL, or about325 mL (inclusive); between about 325 mL and about 10 L, about 9.5 L,about 9 L, about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L,about 6 L, about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L,about 3 L, about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950 mL,about 900 mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL,about 650 mL, about 600 mL, about 550 mL, about 500 mL, about 475 mL,about 450 mL, about 425 mL, about 400 mL, about 375 mL, or about 350 mL(inclusive); between about 350 mL and about 10 L, about 9.5 L, about 9L, about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6L, about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3L, about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950 mL, about900 mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL, about650 mL, about 600 mL, about 550 mL, about 500 mL, about 475 mL, about450 mL, about 425 mL, about 400 mL, or about 375 mL (inclusive); betweenabout 375 mL and about 10 L, about 9.5 L, about 9 L, about 8.5 L, about8 L, about 7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5 L, about5 L, about 4.5 L, about 4 L, about 3.5 L, about 3 L, about 2.5 L, about2 L, about 1.5 L, about 1 L, about 950 mL, about 900 mL, about 850 mL,about 800 mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL,about 550 mL, about 500 mL, about 475 mL, about 450 mL, about 425 mL, orabout 400 mL (inclusive); between about 400 mL and about 10 L, about 9.5L, about 9 L, about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5L, about 6 L, about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5L, about 3 L, about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL, about 700mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL, about 475mL, about 450 mL, or about 425 mL (inclusive); between about 425 mL andabout 10 L, about 9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L,about 7 L, about 6.5 L, about 6 L, about 5.5 L, about 5 L, about 4.5 L,about 4 L, about 3.5 L, about 3 L, about 2.5 L, about 2 L, about 1.5 L,about 1 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL, about750 mL, about 700 mL, about 650 mL, about 600 mL, about 550 mL, about500 mL, about 475 mL, or about 450 mL (inclusive); between about 450 mLand about 10 L, about 9.5 L, about 9 L, about 8.5 L, about 8 L, about7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5 L, about 5 L, about4.5 L, about 4 L, about 3.5 L, about 3 L, about 2.5 L, about 2 L, about1.5 L, about 1 L, about 950 mL, about 900 mL, about 850 mL, about 800mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL, about 550mL, about 500 mL, or about 475 mL (inclusive); between about 475 mL andabout 10 L, about 9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L,about 7 L, about 6.5 L, about 6 L, about 5.5 L, about 5 L, about 4.5 L,about 4 L, about 3.5 L, about 3 L, about 2.5 L, about 2 L, about 1.5 L,about 1 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL, about750 mL, about 700 mL, about 650 mL, about 600 mL, about 550 mL, or about500 mL (inclusive); between about 500 mL and about 10 L, about 9.5 L,about 9 L, about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L,about 6 L, about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L,about 3 L, about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950 mL,about 900 mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL,about 650 mL, about 600 mL, or about 550 mL (inclusive); between about550 mL and about 10 L, about 9.5 L, about 9 L, about 8.5 L, about 8 L,about 7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5 L, about 5 L,about 4.5 L, about 4 L, about 3.5 L, about 3 L, about 2.5 L, about 2 L,about 1.5 L, about 1 L, about 950 mL, about 900 mL, about 850 mL, about800 mL, about 750 mL, about 700 mL, about 650 mL, or about 600 mL(inclusive); between about 600 mL and about 10 L, about 9.5 L, about 9L, about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6L, about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3L, about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950 mL, about900 mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL, or about650 mL (inclusive); between about 650 mL and about 10 L, about 9.5 L,about 9 L, about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L,about 6 L, about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L,about 3 L, about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950 mL,about 900 mL, about 850 mL, about 800 mL, about 750 mL, or about 700 mL(inclusive); between about 700 mL and about 10 L, about 9.5 L, about 9L, about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6L, about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3L, about 2.5 L, about 2 L, about 1.5 L, about 1 L, about 950 mL, about900 mL, about 850 mL, about 800 mL, or about 750 mL (inclusive); betweenabout 750 mL and about 10 L, about 9.5 L, about 9 L, about 8.5 L, about8 L, about 7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5 L, about5 L, about 4.5 L, about 4 L, about 3.5 L, about 3 L, about 2.5 L, about2 L, about 1.5 L, about 1 L, about 950 mL, about 900 mL, about 850 mL,or about 800 mL (inclusive); between about 800 mL and about 10 L, about9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L, about 7 L, about6.5 L, about 6 L, about 5.5 L, about 5 L, about 4.5 L, about 4 L, about3.5 L, about 3 L, about 2.5 L, about 2 L, about 1.5 L, about 1 L, about950 mL, about 900 mL, or about 850 mL (inclusive); between about 850 mLand about 10 L, about 9.5 L, about 9 L, about 8.5 L, about 8 L, about7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5 L, about 5 L, about4.5 L, about 4 L, about 3.5 L, about 3 L, about 2.5 L, about 2 L, about1.5 L, about 1 L, about 950 mL, or about 900 mL (inclusive); betweenabout 900 mL and about 10 L, about 9.5 L, about 9 L, about 8.5 L, about8 L, about 7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5 L, about5 L, about 4.5 L, about 4 L, about 3.5 L, about 3 L, about 2.5 L, about2 L, about 1.5 L, about 1 L, or about 950 mL (inclusive); between about950 mL and about 10 L, about 9.5 L, about 9 L, about 8.5 L, about 8 L,about 7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5 L, about 5 L,about 4.5 L, about 4 L, about 3.5 L, about 3 L, about 2.5 L, about 2 L,about 1.5 L, or about 1 L (inclusive); between about 1 L and about 10 L,about 9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L, about 7 L,about 6.5 L, about 6 L, about 5.5 L, about 5 L, about 4.5 L, about 4 L,about 3.5 L, about 3 L, about 2.5 L, about 2 L, or about 1.5 L(inclusive); between about 1.5 L and about 10 L, about 9.5 L, about 9 L,about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6 L,about 5.5 L, about 5 L, about 4.5 L, about 4 L, about 3.5 L, about 3 L,about 2.5 L, or about 2 L (inclusive); between about 2 L and about 10 L,about 9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L, about 7 L,about 6.5 L, about 6 L, about 5.5 L, about 5 L, about 4.5 L, about 4 L,about 3.5 L, about 3 L, or about 2.5 L (inclusive); between about 2.5 Land about 10 L, about 9.5 L, about 9 L, about 8.5 L, about 8 L, about7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5 L, about 5 L, about4.5 L, about 4 L, about 3.5 L, or about 3 L (inclusive); between about3.0 L and about 10 L, about 9.5 L, about 9 L, about 8.5 L, about 8 L,about 7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5 L, about 5 L,about 4.5 L, about 4 L, or about 3.5 L (inclusive); between about 3.5 Land about 10 L, about 9.5 L, about 9 L, about 8.5 L, about 8 L, about7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5 L, about 5 L, about4.5 L, or about 4 L (inclusive); between about 4 L and about 10 L, about9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L, about 7 L, about6.5 L, about 6 L, about 5.5 L, about 5 L, or about 4.5 L (inclusive);between about 4.5 L and about 10 L, about 9.5 L, about 9 L, about 8.5 L,about 8 L, about 7.5 L, about 7 L, about 6.5 L, about 6 L, about 5.5 L,or about 5 L (inclusive); between about 5 L and about 10 L, about 9.5 L,about 9 L, about 8.5 L, about 8 L, about 7.5 L, about 7 L, about 6.5 L,about 6 L, or about 5.5 L (inclusive); between about 5.5 L and about 10L, about 9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L, about 7L, about 6.5 L, or about 6 L (inclusive); between about 6 L and about 10L, about 9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L, about 7L, or about 6.5 L (inclusive); between about 6.5 L and about 10 L, about9.5 L, about 9 L, about 8.5 L, about 8 L, about 7.5 L, or about 7 L(inclusive); between about 7 L and about 10 L, about 9.5 L, about 9 L,about 8.5 L, about 8 L, or about 7.5 L (inclusive); between about 7.5 Land about 10 L, about 9.5 L, about 9 L, about 8.5 L, or about 8 L(inclusive); between about 8 L and about 10 L, about 9.5 L, about 9 L,or about 8.5 L (inclusive); between about 8.5 L and about 10 L, about9.5 L, or about 9 L (inclusive); between about 9 L and about 10 L orabout 9.5 L (inclusive); or between about 9.5 L and about 10 L.

One or more (e.g., three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, ortwenty-four) different types of buffer can be employed during the use ofthe two or more MCCSs in any of the processes described herein. As isknown in the art, the one or more types of buffer used in the two ormore MCCSs used in the processes described herein will depend on theresin present in the chromatography column(s) and/or the chromatographicmembrane(s) of the two or more MCCSs (e.g., the first and second MCCSs),the recombinant therapeutic protein, and unit operation (e.g., any ofthe exemplary unit operations described herein) performed by thespecific chromatography column(s) and/or chromatography membranes of thetwo or more MCCSs. The volume and type of buffer employed during the useof the two or more MCCSs in any of the processes described herein canalso be determined by one skilled in the art (e.g., discussed in moredetail below). For example, the volume and type(s) of buffer employedduring the use of the two or more MCCSs in any of the processesdescribed herein can be chosen in order to optimize one or more of thefollowing in the recombinant protein drug product: the overall yield ofrecombinant therapeutic protein, the activity of the recombinanttherapeutic protein, the level of purity of the recombinant therapeuticprotein, and the removal of biological contaminants from a fluidcontaining the recombinant therapeutic protein (e.g., absence of activeviruses, mycobacteria, yeast, bacteria, or mammalian cells).

The first and/or second MCCS can be a periodic counter currentchromatography system (PCCS). A PCCS can, e.g., include two or morechromatography columns (e.g., three columns or four columns) that areswitched in order to allow for the continuous elution of recombinanttherapeutic protein from the two or more chromatography columns. A PCCScan include two or more chromatography columns, two or morechromatographic membranes, or at least one chromatographic column and atleast one chromatographic membrane. A column operation generallyconsists of the load, wash, eluate, and regeneration steps. In PCCSs,multiple columns are used to run the same steps discretely andcontinuously in a cyclic fashion. Since the columns are operated inseries, the flow through and wash from one column is captured by anothercolumn. This unique feature of PCCSs allows for loading of the resinclose to its static binding capacity instead of to the dynamic bindingcapacity, as is typical during batch mode chromatography. An example ofthe three column-switching technique used in a PCCS containing threecolumns is shown in FIG. 3. A cycle is defined as three complete columnoperations resulting in an elution pool from each of the three columnsused in the column-switching technique. Once all the steps in the cycleare completed, the cycle is re-started. As a result of the continuouscycling and elutation, fluid entering a PCCS is processed continuously,and the eluate containing recombinant therapeutic protein iscontinuously produced.

To advance from one step to another in a PCCS cycle, such as theexemplary cycle shown in FIG. 3, a column-switching strategy isemployed. The column switching method employs two automated switchingoperations per column in the three-columns in the exemplary PCCS systemshown in FIG. 3, the first of which is related to the initial productbreakthrough, while the second coincides with column saturation. Thedetermination of when the column switching operations should take placeis determined by monitoring the recombinant therapeutic proteinconcentration (e.g., monitoring performed by UV monitoring) in theeluate from each chromatography column present in a PCCS. For example,column switching can be determined by any PAT tool capable of in-linemeasurement of product concentration with feedback control. The PAT toolis capable of real-time in-line measurement of product concentrationwith feedback control.

FIG. 4 depicts an example of column switching in an exemplary PCCS basedon the UV absorbance difference (ΔUV) between the feed inlet and columnoutlet. For example, during column loading (Step 1; FIG. 3), the PCCcontrol system determines the impurity baseline level when theabsorbance stabilizes. As the product breaks through (Step 2; FIG. 3),there is an increase in the outlet UV signal above the impuritybaseline. At the point when ΔUV has reached a pre-determined threshold(e.g., 3% breakthrough of the product), the flow-through from column 1is directed onto column 2 instead of to the waste (t1; FIG. 4). Whencolumn 1 is nearly saturated with product and the ΔUV has reached apre-determined value (t2; FIG. 4), the feed is switched to column 2. Thecolumn-switching strategy used in PCCSs allows for the uniform loadingof the columns irrespective of the feed product concentration and thecapacity. Similar switches of the columns based on the level ofrecombinant protein detected in the eluate from each column can bedesigned. As in known in the art, column switches can also be designedbased on time or the amount of fluid (e.g., buffer) passed through theone or more chromatography column(s) and/or chromatographic membranes inthe first or second MCCS.

In PCCSs, the residence time (RT) of the recombinant therapeutic proteinon the each chromatography column and/or chromatographic membranepresent in the PCCS can be decreased without increasing thecolumn/membrane size because the breakthrough from the firstcolumn/membrane can be captured on another column/membrane in the PCCS.A continuous process system can be designed to process liquid culturemedium at any perfusion rate (D) by varying the column/membrane volume(V) and RT using the equation of: V=D*RT.

The one or more unit operations that can be performed by the at leasttwo MCCSs (e.g., the first and/or second MCCSs) used in the presentlydescribed processes include, for example, capturing the recombinanttherapeutic protein, inactivating viruses present in a fluid containingthe recombinant therapeutic protein, purifying the recombinanttherapeutic protein, polishing the recombinant therapeutic protein,holding a fluid containing the recombinant therapeutic protein (e.g.,using any of the exemplary break tank(s) described herein), filtering orremoving particulate material and/or cells from a fluid containing therecombinant therapeutic protein, and adjusting the ionic concentrationand/or pH of a fluid containing the recombinant therapeutic protein.

The unit operation of capturing can be performed using one or more MCCSs(e.g., a first and/or second MCCS) that includes at least onechromatography column and/or chromatography resin, e.g., that utilizes acapture mechanism. Non-limiting examples of capturing mechanisms includea protein A-binding capture mechanism, an antibody- or antibodyfragment-binding capture mechanism, a substrate-binding capturemechanism, an aptamer-binding capture mechanism, a tag-binding capturemechanism (e.g., poly-His tag-based capture mechanism), and acofactor-binding capture mechanism. Capturing can also be performedusing a resin that can be used to perform cation exchange or anionexchange chromatography, or molecular sieve chromatography. Non-limitingresins that can be used to capture a recombinant therapeutic protein aredescribed herein. Additional examples of resins that can be used tocapture a recombinant therapeutic protein are known in the art.

The unit operation of inactivating viruses present in a fluid containingthe recombinant therapeutic protein can be performed using one or moreMCCSs (e.g., a first and/or second MCCS) that include(s), e.g., achromatography column, a chromatography membrane, or a holding tank thatis capable of incubating a fluid containing the recombinant therapeuticprotein at a pH of between about 3.0 to 5.0 (e.g., between about 3.5 toabout 4.5, between about 3.5 to about 4.25, between about 3.5 to about4.0, between about 3.5 to about 3.8, or about 3.75) for a period of atleast 30 minutes (e.g., a period of between about 30 minutes to 1.5hours, a period of between about 30 minutes to 1.25 hours, a period ofbetween about 0.75 hours to 1.25 hours, or a period of about 1 hour).

The unit operation of purifying a recombinant protein can be performedusing one or more MCCSs (e.g., a first and/or second MCCS) thatinclude(s), e.g., a chromatography column or chromatographic membranethat contains a resin, e.g., that utilizes a capture system.Non-limiting examples of capturing mechanisms include a proteinA-binding capture mechanism, an antibody- or antibody fragment-bindingcapture mechanism, a substrate-binding capture mechanism, anaptamer-binding capture mechanism, a tag-binding capture mechanism(e.g., poly-His tag-based capture mechanism), and a cofactor-bindingcapture mechanism. Purifying can also be performed using a resin thatcan be used to perform cation exchange or anion exchange chromatography,or molecular sieve chromatography. Non-limiting resins that can be usedto purify a recombinant therapeutic protein are described herein.Additional examples of resins that can be used to purify a recombinanttherapeutic protein are known in the art.

The unit operation of polishing a recombinant protein can be performedusing one or more MCCSs (e.g., a first and/or second MCCS) thatinclude(s), e.g., a chromatography column or chromatographic membranethat contains a resin, e.g., that can be used to perform cationexchange, anion exchange, or molecular sieve chromatography.Non-limiting resins that can be used to polish a recombinant therapeuticprotein are described herein. Additional examples of resins that can beused to polish a recombinant therapeutic protein are known in the art.

The unit operation of holding a fluid containing the recombinanttherapeutic protein can be performed using an MCCS (e.g., a first and/orsecond MCCS) that includes at least one reservoir (e.g., a break tank)or a maximum of 1, 2, 3, 4, or 5 reservoir(s) (e.g., break tank(s)) inthe first and second MCCS combined. For example, the reservoir(s) (e.g.,break tank(s)) that can be used to achieve this unit operation can eachhave a volume of, e.g., between about 1 mL to about 10,000 L, about9,000 L, about 8,000 L, about 7,000 L, about 6,000 L, about 5,000 L,about 4,000 L, about 3,000 L, about 2,000 L, about 1,000 L, about 900 L,about 800 L, about 700 L, about 600 L, about 500 L, about 400 L, about300 L, about 200 L, about 100 L, about 80 L, about 60 L, about 40 L,about 20 L, about 10 L, about 5 L, about 1 L, about 900 mL, about 800mL, about 600 mL, about 500 mL, about 400 mL, about 350 mL, about 300mL, about 280 mL, about 260 mL, about 240 mL, about 220 mL, about 200mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL, about 100mL, about 80 mL, about 60 mL, about 40 mL, about 20 mL, or about 10 mL(inclusive); between about 5 mL to about 10,000 L, about 9,000 L, about8,000 L, about 7,000 L, about 6,000 L, about 5,000 L, about 4,000 L,about 3,000 L, about 2,000 L, about 1,000 L, about 900 L, about 800 L,about 700 L, about 600 L, about 500 L, about 400 L, about 300 L, about200 L, about 100 L, about 80 L, about 60 L, about 40 L, about 20 L,about 10 L, about 5 L, about 1 L, about 900 mL, about 800 mL, about 600mL, about 500 mL, about 400 mL, about 350 mL, about 300 mL, about 280mL, about 260 mL, about 240 mL, about 220 mL, about 200 mL, about 180mL, about 160 mL, about 140 mL, about 120 mL, about 100 mL, about 80 mL,about 60 mL, about 40 mL, about 20 mL, or about 10 mL (inclusive);between about 10 mL to about 10,000 L, about 9,000 L, about 8,000 L,about 7,000 L, about 6,000 L, about 5,000 L, about 4,000 L, about 3,000L, about 2,000 L, about 1,000 L, about 900 L, about 800 L, about 700 L,about 600 L, about 500 L, about 400 L, about 300 L, about 200 L, about100 L, about 80 L, about 60 L, about 40 L, about 20 L, about 10 L, about5 L, about 1 L, about 900 mL, about 800 mL, about 600 mL, about 500 mL,about 400 mL, about 350 mL, about 300 mL, about 280 mL, about 260 mL,about 240 mL, about 220 mL, about 200 mL, about 180 mL, about 160 mL,about 140 mL, about 120 mL, about 100 mL, about 80 mL, about 60 mL,about 40 mL, or about 20 mL (inclusive); between about 20 mL to about10,000 L, about 9,000 L, about 8,000 L, about 7,000 L, about 6,000 L,about 5,000 L, about 4,000 L, about 3,000 L, about 2,000 L, about 1,000L, about 900 L, about 800 L, about 700 L, about 600 L, about 500 L,about 400 L, about 300 L, about 200 L, about 100 L, about 80 L, about 60L, about 40 L, about 20 L, about 10 L, about 5 L, about 1 L, about 900mL, about 800 mL, about 600 mL, about 500 mL, about 400 mL, about 350mL, about 300 mL, about 280 mL, about 260 mL, about 240 mL, about 220mL, about 200 mL, about 180 mL, about 160 mL, about 140 mL, about 120mL, about 100 mL, about 80 mL, about 60 mL, or about 40 mL (inclusive);between about 40 mL to about 10,000 L, about 9,000 L, about 8,000 L,about 7,000 L, about 6,000 L, about 5,000 L, about 4,000 L, about 3,000L, about 2,000 L, about 1,000 L, about 900 L, about 800 L, about 700 L,about 600 L, about 500 L, about 400 L, about 300 L, about 200 L, about100 L, about 80 L, about 60 L, about 40 L, about 20 L, about 10 L, about5 L, about 1 L, about 900 mL, about 800 mL, about 600 mL, about 500 mL,about 400 mL, about 350 mL, about 300 mL, about 280 mL, about 260 mL,about 240 mL, about 220 mL, about 200 mL, about 180 mL, about 160 mL,about 140 mL, about 120 mL, about 100 mL, about 80 mL, or about 60 mL(inclusive); between about 60 mL to about 10,000 L, about 9,000 L, about8,000 L, about 7,000 L, about 6,000 L, about 5,000 L, about 4,000 L,about 3,000 L, about 2,000 L, about 1,000 L, about 900 L, about 800 L,about 700 L, about 600 L, about 500 L, about 400 L, about 300 L, about200 L, about 100 L, about 80 L, about 60 L, about 40 L, about 20 L,about 10 L, about 5 L, about 1 L, about 900 mL, about 800 mL, about 600mL, about 500 mL, about 400 mL, about 350 mL, about 300 mL, about 280mL, about 260 mL, about 240 mL, about 220 mL, about 200 mL, about 180mL, about 160 mL, about 140 mL, about 120 mL, about 100 mL, or about 80mL (inclusive); between about 80 mL to about 10,000 L, about 9,000 L,about 8,000 L, about 7,000 L, about 6,000 L, about 5,000 L, about 4,000L, about 3,000 L, about 2,000 L, about 1,000 L, about 900 L, about 800L, about 700 L, about 600 L, about 500 L, about 400 L, about 300 L,about 200 L, about 100 L, about 80 L, about 60 L, about 40 L, about 20L, about 10 L, about 5 L, about 1 L, about 900 mL, about 800 mL, about600 mL, about 500 mL, about 400 mL, about 350 mL, about 300 mL, about280 mL, about 260 mL, about 240 mL, about 220 mL, about 200 mL, about180 mL, about 160 mL, about 140 mL, about 120 mL, or about 100 mL(inclusive); between about 100 mL to about 10,000 L, about 9,000 L,about 8,000 L, about 7,000 L, about 6,000 L, about 5,000 L, about 4,000L, about 3,000 L, about 2,000 L, about 1,000 L, about 900 L, about 800L, about 700 L, about 600 L, about 500 L, about 400 L, about 300 L,about 200 L, about 100 L, about 80 L, about 60 L, about 40 L, about 20L, about 10 L, about 5 L, about 1 L, about 900 mL, about 800 mL, about600 mL, about 500 mL, about 400 mL, about 350 mL, about 300 mL, about280 mL, about 260 mL, about 240 mL, about 220 mL, about 200 mL, about180 mL, about 160 mL, about 140 mL, or about 120 mL (inclusive); betweenabout 120 mL to about 10,000 L, about 9,000 L, about 8,000 L, about7,000 L, about 6,000 L, about 5,000 L, about 4,000 L, about 3,000 L,about 2,000 L, about 1,000 L, about 900 L, about 800 L, about 700 L,about 600 L, about 500 L, about 400 L, about 300 L, about 200 L, about100 L, about 80 L, about 60 L, about 40 L, about 20 L, about 10 L, about5 L, about 1 L, about 900 mL, about 800 mL, about 600 mL, about 500 mL,about 400 mL, about 350 mL, about 300 mL, about 280 mL, about 260 mL,about 240 mL, about 220 mL, about 200 mL, about 180 mL, about 160 mL, orabout 140 mL (inclusive); between about 140 mL to about 10,000 L, about9,000 L, about 8,000 L, about 7,000 L, about 6,000 L, about 5,000 L,about 4,000 L, about 3,000 L, about 2,000 L, about 1,000 L, about 900 L,about 800 L, about 700 L, about 600 L, about 500 L, about 400 L, about300 L, about 200 L, about 100 L, about 80 L, about 60 L, about 40 L,about 20 L, about 10 L, about 5 L, about 1 L, about 900 mL, about 800mL, about 600 mL, about 500 mL, about 400 mL, about 350 mL, about 300mL, about 280 mL, about 260 mL, about 240 mL, about 220 mL, about 200mL, about 180 mL, or about 160 mL (inclusive); between about 160 mL toabout 10,000 L, about 9,000 L, about 8,000 L, about 7,000 L, about 6,000L, about 5,000 L, about 4,000 L, about 3,000 L, about 2,000 L, about1,000 L, about 900 L, about 800 L, about 700 L, about 600 L, about 500L, about 400 L, about 300 L, about 200 L, about 100 L, about 80 L, about60 L, about 40 L, about 20 L, about 10 L, about 5 L, about 1 L, about900 mL, about 800 mL, about 600 mL, about 500 mL, about 400 mL, about350 mL, about 300 mL, about 280 mL, about 260 mL, about 240 mL, about220 mL, about 200 mL, or about 180 mL (inclusive); between about 180 mLto about 10,000 L, about 9,000 L, about 8,000 L, about 7,000 L, about6,000 L, about 5,000 L, about 4,000 L, about 3,000 L, about 2,000 L,about 1,000 L, about 900 L, about 800 L, about 700 L, about 600 L, about500 L, about 400 L, about 300 L, about 200 L, about 100 L, about 80 L,about 60 L, about 40 L, about 20 L, about 10 L, about 5 L, about 1 L,about 900 mL, about 800 mL, about 600 mL, about 500 mL, about 400 mL,about 350 mL, about 300 mL, about 280 mL, about 260 mL, about 240 mL,about 220 mL, or about 200 mL (inclusive); between about 200 mL to about10,000 L, about 9,000 L, about 8,000 L, about 7,000 L, about 6,000 L,about 5,000 L, about 4,000 L, about 3,000 L, about 2,000 L, about 1,000L, about 900 L, about 800 L, about 700 L, about 600 L, about 500 L,about 400 L, about 300 L, about 200 L, about 100 L, about 80 L, about 60L, about 40 L, about 20 L, about 10 L, about 5 L, about 1 L, about 900mL, about 800 mL, about 600 mL, about 500 mL, about 400 mL, about 350mL, about 300 mL, about 280 mL, about 260 mL, about 240 mL, or about 220mL (inclusive); between about 220 mL to about 10,000 L, about 9,000 L,about 8,000 L, about 7,000 L, about 6,000 L, about 5,000 L, about 4,000L, about 3,000 L, about 2,000 L, about 1,000 L, about 900 L, about 800L, about 700 L, about 600 L, about 500 L, about 400 L, about 300 L,about 200 L, about 100 L, about 80 L, about 60 L, about 40 L, about 20L, about 10 L, about 5 L, about 1 L, about 900 mL, about 800 mL, about600 mL, about 500 mL, about 400 mL, about 350 mL, about 300 mL, about280 mL, about 260 mL, or about 240 mL (inclusive); between about 240 mLto about 10,000 L, about 9,000 L, about 8,000 L, about 7,000 L, about6,000 L, about 5,000 L, about 4,000 L, about 3,000 L, about 2,000 L,about 1,000 L, about 900 L, about 800 L, about 700 L, about 600 L, about500 L, about 400 L, about 300 L, about 200 L, about 100 L, about 80 L,about 60 L, about 40 L, about 20 L, about 10 L, about 5 L, about 1 L,about 900 mL, about 800 mL, about 600 mL, about 500 mL, about 400 mL,about 350 mL, about 300 mL, about 280 mL, or about 260 mL (inclusive);about 260 mL to about 10,000 L, about 9,000 L, about 8,000 L, about7,000 L, about 6,000 L, about 5,000 L, about 4,000 L, about 3,000 L,about 2,000 L, about 1,000 L, about 900 L, about 800 L, about 700 L,about 600 L, about 500 L, about 400 L, about 300 L, about 200 L, about100 L, about 80 L, about 60 L, about 40 L, about 20 L, about 10 L, about5 L, about 1 L, about 900 mL, about 800 mL, about 600 mL, about 500 mL,about 400 mL, about 350 mL, about 300 mL, or about 280 mL (inclusive);about 280 mL to about 10,000 L, about 9,000 L, about 8,000 L, about7,000 L, about 6,000 L, about 5,000 L, about 4,000 L, about 3,000 L,about 2,000 L, about 1,000 L, about 900 L, about 800 L, about 700 L,about 600 L, about 500 L, about 400 L, about 300 L, about 200 L, about100 L, about 80 L, about 60 L, about 40 L, about 20 L, about 10 L, about5 L, about 1 L, about 900 mL, about 800 mL, about 600 mL, about 500 mL,about 400 mL, about 350 mL, or about 300 mL (inclusive); between about300 mL to about 10,000 L, about 9,000 L, about 8,000 L, about 7,000 L,about 6,000 L, about 5,000 L, about 4,000 L, about 3,000 L, about 2,000L, about 1,000 L, about 900 L, about 800 L, about 700 L, about 600 L,about 500 L, about 400 L, about 300 L, about 200 L, about 100 L, about80 L, about 60 L, about 40 L, about 20 L, about 10 L, about 5 L, about 1L, about 900 mL, about 800 mL, about 600 mL, about 500 mL, about 400 mL,or about 350 mL (inclusive); between about 350 mL to about 10,000 L,about 9,000 L, about 8,000 L, about 7,000 L, about 6,000 L, about 5,000L, about 4,000 L, about 3,000 L, about 2,000 L, about 1,000 L, about 900L, about 800 L, about 700 L, about 600 L, about 500 L, about 400 L,about 300 L, about 200 L, about 100 L, about 80 L, about 60 L, about 40L, about 20 L, about 10 L, about 5 L, about 1 L, about 900 mL, about 800mL, about 600 mL, about 500 mL, or about 400 mL (inclusive); betweenabout 400 mL to about 10,000 L, about 9,000 L, about 8,000 L, about7,000 L, about 6,000 L, about 5,000 L, about 4,000 L, about 3,000 L,about 2,000 L, about 1,000 L, about 900 L, about 800 L, about 700 L,about 600 L, about 500 L, about 400 L, about 300 L, about 200 L, about100 L, about 80 L, about 60 L, about 40 L, about 20 L, about 10 L, about5 L, about 1 L, about 900 mL, about 800 mL, about 600 mL, or about 500mL (inclusive); between about 500 mL to about 10,000 L, about 9,000 L,about 8,000 L, about 7,000 L, about 6,000 L, about 5,000 L, about 4,000L, about 3,000 L, about 2,000 L, about 1,000 L, about 900 L, about 800L, about 700 L, about 600 L, about 500 L, about 400 L, about 300 L,about 200 L, about 100 L, about 80 L, about 60 L, about 40 L, about 20L, about 10 L, about 5 L, about 1 L, about 900 mL, about 800 mL, orabout 600 mL (inclusive); between about 600 mL to about 10,000 L, about9,000 L, about 8,000 L, about 7,000 L, about 6,000 L, about 5,000 L,about 4,000 L, about 3,000 L, about 2,000 L, about 1,000 L, about 900 L,about 800 L, about 700 L, about 600 L, about 500 L, about 400 L, about300 L, about 200 L, about 100 L, about 80 L, about 60 L, about 40 L,about 20 L, about 10 L, about 5 L, about 1 L, about 900 mL, or about 800mL (inclusive); between about 800 mL to about 10,000 L, about 9,000 L,about 8,000 L, about 7,000 L, about 6,000 L, about 5,000 L, about 4,000L, about 3,000 L, about 2,000 L, about 1,000 L, about 900 L, about 800L, about 700 L, about 600 L, about 500 L, about 400 L, about 300 L,about 200 L, about 100 L, about 80 L, about 60 L, about 40 L, about 20L, about 10 L, about 5 L, about 1 L, or about 900 mL (inclusive);between about 900 mL to about 10,000 L, about 9,000 L, about 8,000 L,about 7,000 L, about 6,000 L, about 5,000 L, about 4,000 L, about 3,000L, about 2,000 L, about 1,000 L, about 900 L, about 800 L, about 700 L,about 600 L, about 500 L, about 400 L, about 300 L, about 200 L, about100 L, about 80 L, about 60 L, about 40 L, about 20 L, about 10 L, about5 L, or about 1 L (inclusive); between about 1 L to about 10,000 L,about 9,000 L, about 8,000 L, about 7,000 L, about 6,000 L, about 5,000L, about 4,000 L, about 3,000 L, about 2,000 L, about 1,000 L, about 900L, about 800 L, about 700 L, about 600 L, about 500 L, about 400 L,about 300 L, about 200 L, about 100 L, about 80 L, about 60 L, about 40L, about 20 L, about 10 L, or about 5 L (inclusive); between about 5 Lto about 10,000 L, about 9,000 L, about 8,000 L, about 7,000 L, about6,000 L, about 5,000 L, about 4,000 L, about 3,000 L, about 2,000 L,about 1,000 L, about 900 L, about 800 L, about 700 L, about 600 L, about500 L, about 400 L, about 300 L, about 200 L, about 100 L, about 80 L,about 60 L, about 40 L, about 20 L, or about 10 L (inclusive); betweenabout 10 L to about 10,000 L, about 9,000 L, about 8,000 L, about 7,000L, about 6,000 L, about 5,000 L, about 4,000 L, about 3,000 L, about2,000 L, about 1,000 L, about 900 L, about 800 L, about 700 L, about 600L, about 500 L, about 400 L, about 300 L, about 200 L, about 100 L,about 80 L, about 60 L, about 40 L, or about 20 L (inclusive); betweenabout 20 L to about 10,000 L, about 9,000 L, about 8,000 L, about 7,000L, about 6,000 L, about 5,000 L, about 4,000 L, about 3,000 L, about2,000 L, about 1,000 L, about 900 L, about 800 L, about 700 L, about 600L, about 500 L, about 400 L, about 300 L, about 200 L, about 100 L,about 80 L, about 60 L, or about 40 L (inclusive); between about 40 L toabout 10,000 L, about 9,000 L, about 8,000 L, about 7,000 L, about 6,000L, about 5,000 L, about 4,000 L, about 3,000 L, about 2,000 L, about1,000 L, about 900 L, about 800 L, about 700 L, about 600 L, about 500L, about 400 L, about 300 L, about 200 L, about 100 L, about 80 L, orabout 60 L (inclusive); between about 60 L to about 10,000 L, about9,000 L, about 8,000 L, about 7,000 L, about 6,000 L, about 5,000 L,about 4,000 L, about 3,000 L, about 2,000 L, about 1,000 L, about 900 L,about 800 L, about 700 L, about 600 L, about 500 L, about 400 L, about300 L, about 200 L, about 100 L, or about 80 L (inclusive); betweenabout 80 L to about 10,000 L, about 9,000 L, about 8,000 L, about 7,000L, about 6,000 L, about 5,000 L, about 4,000 L, about 3,000 L, about2,000 L, about 1,000 L, about 900 L, about 800 L, about 700 L, about 600L, about 500 L, about 400 L, about 300 L, about 200 L, or about 100 L(inclusive); between about 100 L to about 10,000 L, about 9,000 L, about8,000 L, about 7,000 L, about 6,000 L, about 5,000 L, about 4,000 L,about 3,000 L, about 2,000 L, about 1,000 L, about 900 L, about 800 L,about 700 L, about 600 L, about 500 L, about 400 L, about 300 L, orabout 200 L (inclusive); between about 200 L to about 10,000 L, about9,000 L, about 8,000 L, about 7,000 L, about 6,000 L, about 5,000 L,about 4,000 L, about 3,000 L, about 2,000 L, about 1,000 L, about 900 L,about 800 L, about 700 L, about 600 L, about 500 L, about 400 L, orabout 300 L (inclusive); between about 300 L to about 10,000 L, about9,000 L, about 8,000 L, about 7,000 L, about 6,000 L, about 5,000 L,about 4,000 L, about 3,000 L, about 2,000 L, about 1,000 L, about 900 L,about 800 L, about 700 L, about 600 L, about 500 L, or about 400 L(inclusive); between about 400 L to about 10,000 L, about 9,000 L, about8,000 L, about 7,000 L, about 6,000 L, about 5,000 L, about 4,000 L,about 3,000 L, about 2,000 L, about 1,000 L, about 900 L, about 800 L,about 700 L, about 600 L, or about 500 L (inclusive); between about 500L to about 10,000 L, about 9,000 L, about 8,000 L, about 7,000 L, about6,000 L, about 5,000 L, about 4,000 L, about 3,000 L, about 2,000 L,about 1,000 L, about 900 L, about 800 L, about 700 L, or about 600 L(inclusive); between about 600 L to about 10,000 L, about 9,000 L, about8,000 L, about 7,000 L, about 6,000 L, about 5,000 L, about 4,000 L,about 3,000 L, about 2,000 L, about 1,000 L, about 900 L, about 800 L,or about 700 L (inclusive); between about 700 L to about 10,000 L, about9,000 L, about 8,000 L, about 7,000 L, about 6,000 L, about 5,000 L,about 4,000 L, about 3,000 L, about 2,000 L, about 1,000 L, about 900 L,or about 800 L (inclusive); between about 800 L to about 10,000 L, about9,000 L, about 8,000 L, about 7,000 L, about 6,000 L, about 5,000 L,about 4,000 L, about 3,000 L, about 2,000 L, about 1,000 L, or about 900L (inclusive); between about 900 L to about 10,000 L, about 9,000 L,about 8,000 L, about 7,000 L, about 6,000 L, about 5,000 L, about 4,000L, about 3,000 L, about 2,000 L, or about 1,000 L (inclusive); betweenabout 1,000 L to about 10,000 L, about 9,000 L, about 8,000 L, about7,000 L, about 6,000 L, about 5,000 L, about 4,000 L, about 3,000 L, orabout 2,000 L (inclusive); between about 2,000 L to about 10,000 L,about 9,000 L, about 8,000 L, about 7,000 L, about 6,000 L, about 5,000L, about 4,000 L, or about 3,000 L (inclusive); between about 3,000 L toabout 10,000 L, about 9,000 L, about 8,000 L, about 7,000 L, about 6,000L, about 5,000 L, or about 4,000 L (inclusive); between about 4,000 L toabout 10,000 L, about 9,000 L, about 8,000 L, about 7,000 L, about 6,000L, or about 5,000 L (inclusive); between about 5,000 L to about 10,000L, about 9,000 L, about 8,000 L, about 7,000 L, or about 6,000 L(inclusive); between about 6,000 L to about 10,000 L, about 9,000 L,about 8,000 L, or about 7,000 L (inclusive); between about 7,000 L toabout 10,000 L, about 9,000 L, or about 8,000 L (inclusive); betweenabout 8,000 L to about 10,000 L or about 9,000 L (inclusive); or betweenabout 9,000 L to about 10,000 L (inclusive).

The reservoir(s) (e.g., break tank(s)) used in the processes describedherein can have a capacity that is, e.g., between about 1 mL to about10,000 L, inclusive (or any of the subranges of this range of volumesdescribed herein). Any of the reservoir(s) (e.g., break tank(s)) used(in any of the processes described herein) to hold fluid before itenters into the first MCCS can have a capacity that is, e.g., between 1mL and about 100%, inclusive, between about 1 mL and about 90%, about80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%,about 10%, or about 5%, inclusive, of the loading volume of the firstcolumn of the first MCCS. Any of the reservoir(s) (e.g., break tanks(s))used to hold a fluid before it enters the second MCCS (and after thefluid leaves the first MCCS) can have a capacity that is, e.g., between1 mL and about 100%, inclusive, e.g., between about 1 mL and about 90%,about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about20%, about 10%, or about 5%, inclusive, of the loading volume of thefirst column of the second MCCS.

The reservoir(s) (e.g., break tank(s)) can each hold the fluidcontaining the recombinant therapeutic protein for at least 10 minutes(e.g., at least 20 minutes, at least 30 minutes, at least 1 hour, atleast 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, atleast 10 hours, at least 12 hours, at least 14 hours, at least 16 hours,at least 18 hours, at least 20 hours, at least 22 hours, at least 24hours, at least 26 hours, at least 28 hours, at least 30 hours, at least32 hours, at least 34 hours, at least 36 hours, at least 38 hours, atleast 40 hours, at least 42 hours, at least 44 hours, at least 46 hours,or at least 48 hours). In other examples, the reservoir(s) (e.g., breaktank(s)) only holds a therapeutic protein for a total time period of,e.g., between about 5 minutes and less than about 48 hours, inclusive,e.g., between about 5 minutes and about 48 hours, about 46 hours, about44 hours, about 42 hours, about 40 hours, about 38 hours, about 36hours, about 34 hours, about 32 hours, about 30 hours, about 28 hours,about 26 hours, about 24 hours, about 22 hours, about 20 hours, about 18hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours,about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, orabout 30 minutes, inclusive; between about 30 minutes and about 48hours, about 46 hours, about 44 hours, about 42 hours, about 40 hours,about 38 hours, about 36 hours, about 34 hours, about 32 hours, about 30hours, about 28 hours, about 26 hours, about 24 hours, about 22 hours,about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours,about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2hours, or about 1 hour, inclusive; between about 1 hour and about 48hours, about 46 hours, about 44 hours, about 42 hours, about 40 hours,about 38 hours, about 36 hours, about 34 hours, about 32 hours, about 30hours, about 28 hours, about 26 hours, about 24 hours, about 22 hours,about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours,about 6 hours, about 5 hours, about 4 hours, about 3 hours, or about 2hours, inclusive; between about 2 hours and about 48 hours, about 46hours, about 44 hours, about 42 hours, about 40 hours, about 38 hours,about 36 hours, about 34 hours, about 32 hours, about 30 hours, about 28hours, about 26 hours, about 24 hours, about 22 hours, about 20 hours,about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about5 hours, about 4 hours, or about 3 hours, inclusive; between about 3hours and about 48 hours, about 46 hours, about 44 hours, about 42hours, about 40 hours, about 38 hours, about 36 hours, about 34 hours,about 32 hours, about 30 hours, about 28 hours, about 26 hours, about 24hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours,about 14 hours, about 12 hours, about 10 hours, about 9 hours, about 8hours, about 7 hours, about 6 hours, about 5 hours, or about 4 hours,inclusive; between about 4 hours and about 48 hours, about 46 hours,about 44 hours, about 42 hours, about 40 hours, about 38 hours, about 36hours, about 34 hours, about 32 hours, about 30 hours, about 28 hours,about 26 hours, about 24 hours, about 22 hours, about 20 hours, about 18hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours,about 9 hours, about 8 hours, about 7 hours, about 6 hours, or about 5hours, inclusive; between about 5 hours and about 48 hours, about 46hours, about 44 hours, about 42 hours, about 40 hours, about 38 hours,about 36 hours, about 34 hours, about 32 hours, about 30 hours, about 28hours, about 26 hours, about 24 hours, about 22 hours, about 20 hours,about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10hours, about 9 hours, about 8 hours, about 7 hours, or about 6 hours,inclusive; between about 6 hours and about 48 hours, about 46 hours,about 44 hours, about 42 hours, about 40 hours, about 38 hours, about 36hours, about 34 hours, about 32 hours, about 30 hours, about 28 hours,about 26 hours, about 24 hours, about 22 hours, about 20 hours, about 18hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours,about 9 hours, about 8 hours, or about 7 hours, inclusive; between about7 hours and about 48 hours, about 46 hours, about 44 hours, about 42hours, about 40 hours, about 38 hours, about 36 hours, about 34 hours,about 32 hours, about 30 hours, about 28 hours, about 26 hours, about 24hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours,about 14 hours, about 12 hours, about 10 hours, about 9 hours, or about8 hours, inclusive; between about 8 hours and about 48 hours, about 46hours, about 44 hours, about 42 hours, about 40 hours, about 38 hours,about 36 hours, about 34 hours, about 32 hours, about 30 hours, about 28hours, about 26 hours, about 24 hours, about 22 hours, about 20 hours,about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10hours, or about 9 hours, inclusive; between about 9 hours and about 48hours, about 46 hours, about 44 hours, about 42 hours, about 40 hours,about 38 hours, about 36 hours, about 34 hours, about 32 hours, about 30hours, about 28 hours, about 26 hours, about 24 hours, about 22 hours,about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12hours, or about 10 hours, inclusive; between about 10 hours and about 48hours, about 46 hours, about 44 hours, about 42 hours, about 40 hours,about 38 hours, about 36 hours, about 34 hours, about 32 hours, about 30hours, about 28 hours, about 26 hours, about 24 hours, about 22 hours,about 20 hours, about 18 hours, about 16 hours, about 14 hours, or about12 hours, inclusive; between about 12 hours and about 48 hours, about 46hours, about 44 hours, about 42 hours, about 40 hours, about 38 hours,about 36 hours, about 34 hours, about 32 hours, about 30 hours, about 28hours, about 26 hours, about 24 hours, about 22 hours, about 20 hours,about 18 hours, about 16 hours, or about 14 hours, inclusive; betweenabout 14 hours and about 48 hours, about 46 hours, about 44 hours, about42 hours, about 40 hours, about 38 hours, about 36 hours, about 34hours, about 32 hours, about 30 hours, about 28 hours, about 26 hours,about 24 hours, about 22 hours, about 20 hours, about 18 hours, or about16 hours, inclusive; between about 16 hours and about 48 hours, about 46hours, about 44 hours, about 42 hours, about 40 hours, about 38 hours,about 36 hours, about 34 hours, about 32 hours, about 30 hours, about 28hours, about 26 hours, about 24 hours, about 22 hours, about 20 hours,or about 18 hours, inclusive; between about 18 hours and about 48 hours,about 46 hours, about 44 hours, about 42 hours, about 40 hours, about 38hours, about 36 hours, about 34 hours, about 32 hours, about 30 hours,about 28 hours, about 26 hours, about 24 hours, about 22 hours, or about20 hours, inclusive; between about 20 hours and about 48 hours, about 46hours, about 44 hours, about 42 hours, about 40 hours, about 38 hours,about 36 hours, about 34 hours, about 32 hours, about 30 hours, about 28hours, about 26 hours, about 24 hours, or about 22 hours, inclusive;between about 22 hours and about 48 hours, about 46 hours, about 44hours, about 42 hours, about 40 hours, about 38 hours, about 36 hours,about 34 hours, about 32 hours, about 30 hours, about 28 hours, about 26hours, or about 24 hours, inclusive; between about 24 hours and about 48hours, about 46 hours, about 44 hours, about 42 hours, about 40 hours,about 38 hours, about 36 hours, about 34 hours, about 32 hours, about 30hours, about 28 hours, or about 26 hours, inclusive; between about 26hours and about 48 hours, about 46 hours, about 44 hours, about 42hours, about 40 hours, about 38 hours, about 36 hours, about 34 hours,about 32 hours, about 30 hours, or about 28 hours, inclusive; betweenabout 28 hours and about 48 hours, about 46 hours, about 44 hours, about42 hours, about 40 hours, about 38 hours, about 36 hours, about 34hours, about 32 hours, or about 30 hours, inclusive; between about 30hours and about 48 hours, about 46 hours, about 44 hours, about 42hours, about 40 hours, about 38 hours, about 36 hours, about 34 hours,or about 32 hours, inclusive; between about 32 hours and about 48 hours,about 46 hours, about 44 hours, about 42 hours, about 40 hours, about 38hours, about 36 hours, or about 34 hours, inclusive; between about 34hours and about 48 hours, about 46 hours, about 44 hours, about 42hours, about 40 hours, about 38 hours, or about 36 hours, inclusive;between about 36 hours and about 48 hours, about 46 hours, about 44hours, about 42 hours, about 40 hours, or about 38 hours, inclusive;between about 38 hours and about 48 hours, about 46 hours, about 44hours, about 42 hours, or about 40 hours, inclusive; between about 40hours and about 48 hours, about 46 hours, about 44 hours, or about 42hours, inclusive; between about 42 hours and about 48 hours, about 46hours, or about 44 hours, inclusive; between about 44 hours and about 48hours or about 46 hours, inclusive; between about 46 hours and about 48hours, inclusive.

The reservoir(s) (e.g., break tank(s)) can be used to both hold andrefrigerate (e.g., at a temperature of less than 25° C., less than 15°C., or less than 10° C.) the fluid containing the recombinanttherapeutic protein. The reservoir can have any shape, including acircular cylinder, an oval cylinder, or an approximately rectangularsealed and nonpermeable bag.

The unit operations of filtering a fluid containing the recombinanttherapeutic protein can be performed using an MCCS (e.g., the firstand/or second MCCS) that includes, e.g., a filter, or a chromatographycolumn or chromatographic membrane that contains a molecule sieve resin.As is known in the art, a wide variety of submicron filters (e.g., afilter with a pore size of less than 1 μm, less than 0.5 μm, less than0.3 μm, about 0.2 μm, less than 0.2 μm, less than 100 nm, less than 80nm, less than 60 nm, less than 40 nm, less than 20 nm, or less than 10nm) are available in the art that are capable of removing anyprecipitated material and/or cells (e.g., precipitated, unfoldedprotein; precipitated, unwanted host cell proteins; precipitated lipids;bacteria; yeast cells; fungal cells; mycobacteria; and/or mammaliancells). Filters having a pore size of about 0.2 μm or less than 0.2 μmare known to effectively remove bacteria from the fluid containing therecombinant therapeutic protein. As is known in the art, achromatography column or a chromatographic membrane containing amolecular sieve resin can also be used in an MCCS (e.g., the firstand/or second MCCS) to perform the unit operation of filtering a fluidcontaining a recombinant therapeutic protein.

The unit operations of adjusting the ionic concentration and/or pH of afluid containing the recombinant therapeutic protein can be performedusing a MCCS (e.g., the first and/or second MCCS) that includes andutilizes a buffer adjustment reservoir (e.g., an in-line bufferadjustment reservoir) that adds a new buffer solution into a fluid thatcontains the recombinant therapeutic protein (e.g., between columnswithin a single MCCS, or after the last column in a penultimate MCCS(e.g., the first MCCS) and before the fluid containing the recombinanttherapeutic protein is fed into the first column of the next MCCS (e.g.,the second MCCS). As can be appreciated in the art, the in-line bufferadjustment reservoir can be any size (e.g., greater than 100 mL) and cancontain any buffered solution (e.g., a buffered solution that has one ormore of: an increased or decreased pH as compared to the fluidcontaining the recombinant therapeutic protein, a an increased ordecreased ionic (e.g., salt) concentration compared to the fluidcontaining the recombinant therapeutic protein, and/or an increased ordecreased concentration of an agent that competes with the recombinanttherapeutic protein for binding to resin present in at least onechromatographic column or at least one chromatographic membrane in anMCCS (e.g., the first or the second MCCS)). In other examples, the unitoperations of adjusting the ionic concentration and/or pH of a fluid canbe performed using size exclusion chromatography and counter-currentdialysis (see, e.g., Kurnik et al., Biotechnol. Bioeng. 45(2):149-157,1995; and Klutz et al., J. Biotechnol. 213:120-130, 2015).

The first and/or second MCCS can perform two or more unit operations.For example, the first and/or second MCCS can each perform at least thefollowing unit operations: capturing the recombinant therapeutic proteinand inactivating viruses present in the fluid containing the recombinanttherapeutic protein; capturing the recombinant therapeutic protein,inactivating viruses present in the fluid containing the recombinanttherapeutic protein, and adjusting the ionic concentration and/or pH ofa liquid containing the recombinanant therapeutic protein; purifying therecombinant therapeutic protein and polishing the recombinanttherapeutic protein; purifying the recombinant therapeutic protein,polishing the recombinant therapeutic protein, and filtering a fluidcontaining the recombinant therapeutic protein or removing precipitatesand/or particular matter from a fluid containing the recombinanttherapeutic protein; and purifying the recombinant therapeutic protein,polishing the recombinant therapeutic protein, filtering a fluidcontaining the recombinant therapeutic protein or removing precipitatesand/or particular matter from a fluid containing the recombinanttherapeutic protein, and adjusting the ionic concentration and/or pH ofa liquid containing the recombinant therapeutic protein.

Capturing the Recombinant Therapeutic Protein

The present processes include a step of capturing the recombinanttherapeutic protein using a first MCCS. As can be appreciated in theart, the liquid culture medium containing the recombinant therapeuticprotein can be continuously fed onto the first MCCS using a variety ofdifferent means. For example, the liquid culture medium can be activelypumped into the first MCCS or the liquid culture medium can be fed intothe first MCCS using gravitational force. The liquid culture medium canbe stored in a reservoir (e.g., a holding tank) before it is fed intothe first MCCS or the liquid culture medium can be actively pumped froma bioreactor containing a culture of cells (e.g., mammalian cells thatsecrete the recombinant therapeutic protein into the culture medium)into the first MCCS.

The liquid culture medium can be fed (loaded) into the first MCCS at aflow rate of, e.g., between about 0.2 mL/minute to about 100 L/minute,about 95 L/minute, about 90 L/minute, about 85 L/minute, about 80L/minute, about 75 L/minute, about 70 L/minute, about 65 L/minute, about60 L/minute, about 55 L/minute, about 50 L/minute, about 45 L/minute,about 40 L/minute, about 35 L/minute, about 30 L/minute, about 25L/minute, about 20 L/minute, about 15 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 95 mL/minute, about 90 mL/minute, about 85 mL/minute,about 80 mL/minute, about 75 mL/minute, about 70 mL/minute, about 65mL/minute, about 60 mL/minute, about 55 mL/minute, about 50 mL/minute,about 45 mL/minute, about 40 mL/minute, about 35 mL/minute, about 30mL/minute, about 25 mL/minute, about 20 mL/minute, about 15 mL/minute,about 10 mL/minute, about 5 mL/minute, or about 2 mL/minute (inclusive);between about 0.5 mL/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute, about55 L/minute, about 50 L/minute, about 45 L/minute, about 40 L/minute,about 35 L/minute, about 30 L/minute, about 25 L/minute, about 20L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 95 mL/minute, about 90 mL/minute, about 85 mL/minute,about 80 mL/minute, about 75 mL/minute, about 70 mL/minute, about 65mL/minute, about 60 mL/minute, about 55 mL/minute, about 50 mL/minute,about 45 mL/minute, about 40 mL/minute, about 35 mL/minute, about 30mL/minute, about 25 mL/minute, about 20 mL/minute, about 15 mL/minute,about 10 mL/minute, about 5 mL/minute, or about 2 mL/minute (inclusive);between about 1 mL/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute, about55 L/minute, about 50 L/minute, about 45 L/minute, about 40 L/minute,about 35 L/minute, about 30 L/minute, about 25 L/minute, about 20L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 95 mL/minute, about 90 mL/minute, about 85 mL/minute,about 80 mL/minute, about 75 mL/minute, about 70 mL/minute, about 65mL/minute, about 60 mL/minute, about 55 mL/minute, about 50 mL/minute,about 45 mL/minute, about 40 mL/minute, about 35 mL/minute, about 30mL/minute, about 25 mL/minute, about 20 mL/minute, about 15 mL/minute,about 10 mL/minute, about 5 mL/minute, or about 2 mL/minute (inclusive);between about 2 mL/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute, about55 L/minute, about 50 L/minute, about 45 L/minute, about 40 L/minute,about 35 L/minute, about 30 L/minute, about 25 L/minute, about 20L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 95 mL/minute, about 90 mL/minute, about 85 mL/minute,about 80 mL/minute, about 75 mL/minute, about 70 mL/minute, about 65mL/minute, about 60 mL/minute, about 55 mL/minute, about 50 mL/minute,about 45 mL/minute, about 40 mL/minute, about 35 mL/minute, about 30mL/minute, about 25 mL/minute, about 20 mL/minute, about 15 mL/minute,about 10 mL/minute, or about 5 mL/minute (inclusive); between about 5mL/minute to about 100 L/minute, about 95 L/minute, about 90 L/minute,about 85 L/minute, about 80 L/minute, about 75 L/minute, about 70L/minute, about 65 L/minute, about 60 L/minute, about 55 L/minute, about50 L/minute, about 45 L/minute, about 40 L/minute, about 35 L/minute,about 30 L/minute, about 25 L/minute, about 20 L/minute, about 15L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about950 mL/minute, about 900 mL/minute, about 850 mL/minute, about 800mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 95 mL/minute,about 90 mL/minute, about 85 mL/minute, about 80 mL/minute, about 75mL/minute, about 70 mL/minute, about 65 mL/minute, about 60 mL/minute,about 55 mL/minute, about 50 mL/minute, about 45 mL/minute, about 40mL/minute, about 35 mL/minute, about 30 mL/minute, about 25 mL/minute,about 20 mL/minute, about 15 mL/minute, or about 10 mL/minute(inclusive); between 10 mL/minute to about 100 L/minute, about 95L/minute, about 90 L/minute, about 85 L/minute, about 80 L/minute, about75 L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute,about 55 L/minute, about 50 L/minute, about 45 L/minute, about 40L/minute, about 35 L/minute, about 30 L/minute, about 25 L/minute, about20 L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute,about 1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 95 mL/minute, about 90 mL/minute, about 85 mL/minute,about 80 mL/minute, about 75 mL/minute, about 70 mL/minute, about 65mL/minute, about 60 mL/minute, about 55 mL/minute, about 50 mL/minute,about 45 mL/minute, about 40 mL/minute, about 35 mL/minute, about 30mL/minute, about 25 mL/minute, about 20 mL/minute, or about 15 mL/minute(inclusive); between about 15 mL/minute to about 100 L/minute, about 95L/minute, about 90 L/minute, about 85 L/minute, about 80 L/minute, about75 L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute,about 55 L/minute, about 50 L/minute, about 45 L/minute, about 40L/minute, about 35 L/minute, about 30 L/minute, about 25 L/minute, about20 L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute,about 1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 95 mL/minute, about 90 mL/minute, about 85 mL/minute,about 80 mL/minute, about 75 mL/minute, about 70 mL/minute, about 65mL/minute, about 60 mL/minute, about 55 mL/minute, about 50 mL/minute,about 45 mL/minute, about 40 mL/minute, about 35 mL/minute, about 30mL/minute, about 25 mL/minute, or about 20 mL/minute (inclusive);between about 20 mL/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute, about55 L/minute, about 50 L/minute, about 45 L/minute, about 40 L/minute,about 35 L/minute, about 30 L/minute, about 25 L/minute, about 20L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 95 mL/minute, about 90 mL/minute, about 85 mL/minute,about 80 mL/minute, about 75 mL/minute, about 70 mL/minute, about 65mL/minute, about 60 mL/minute, about 55 mL/minute, about 50 mL/minute,about 45 mL/minute, about 40 mL/minute, about 35 mL/minute, about 30mL/minute, or about 25 mL/minute (inclusive); between about 25 mL/minuteto about 100 L/minute, about 95 L/minute, about 90 L/minute, about 85L/minute, about 80 L/minute, about 75 L/minute, about 70 L/minute, about65 L/minute, about 60 L/minute, about 55 L/minute, about 50 L/minute,about 45 L/minute, about 40 L/minute, about 35 L/minute, about 30L/minute, about 25 L/minute, about 20 L/minute, about 15 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 95 mL/minute, about 90 mL/minute,about 85 mL/minute, about 80 mL/minute, about 75 mL/minute, about 70mL/minute, about 65 mL/minute, about 60 mL/minute, about 55 mL/minute,about 50 mL/minute, about 45 mL/minute, about 40 mL/minute, about 35mL/minute, or about 30 mL/minute (inclusive); between about 30 mL/minuteto about 100 L/minute, about 95 L/minute, about 90 L/minute, about 85L/minute, about 80 L/minute, about 75 L/minute, about 70 L/minute, about65 L/minute, about 60 L/minute, about 55 L/minute, about 50 L/minute,about 45 L/minute, about 40 L/minute, about 35 L/minute, about 30L/minute, about 25 L/minute, about 20 L/minute, about 15 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 95 mL/minute, about 90 mL/minute,about 85 mL/minute, about 80 mL/minute, about 75 mL/minute, about 70mL/minute, about 65 mL/minute, about 60 mL/minute, about 55 mL/minute,about 50 mL/minute, about 45 mL/minute, about 40 mL/minute, or about 35mL/minute (inclusive); between about 35 mL/minute to about 100 L/minute,about 95 L/minute, about 90 L/minute, about 85 L/minute, about 80L/minute, about 75 L/minute, about 70 L/minute, about 65 L/minute, about60 L/minute, about 55 L/minute, about 50 L/minute, about 45 L/minute,about 40 L/minute, about 35 L/minute, about 30 L/minute, about 25L/minute, about 20 L/minute, about 15 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 95 mL/minute, about 90 mL/minute, about 85 mL/minute,about 80 mL/minute, about 75 mL/minute, about 70 mL/minute, about 65mL/minute, about 60 mL/minute, about 55 mL/minute, about 50 mL/minute,about 45 mL/minute, or about 40 mL/minute (inclusive); between about 40mL/minute to about 100 L/minute, about 95 L/minute, about 90 L/minute,about 85 L/minute, about 80 L/minute, about 75 L/minute, about 70L/minute, about 65 L/minute, about 60 L/minute, about 55 L/minute, about50 L/minute, about 45 L/minute, about 40 L/minute, about 35 L/minute,about 30 L/minute, about 25 L/minute, about 20 L/minute, about 15L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about950 mL/minute, about 900 mL/minute, about 850 mL/minute, about 800mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 95 mL/minute,about 90 mL/minute, about 85 mL/minute, about 80 mL/minute, about 75mL/minute, about 70 mL/minute, about 65 mL/minute, about 60 mL/minute,about 55 mL/minute, about 50 mL/minute, or about 45 mL/minute(inclusive); between about 45 mL/minute to about 100 L/minute, about 95L/minute, about 90 L/minute, about 85 L/minute, about 80 L/minute, about75 L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute,about 55 L/minute, about 50 L/minute, about 45 L/minute, about 40L/minute, about 35 L/minute, about 30 L/minute, about 25 L/minute, about20 L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute,about 1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 95 mL/minute, about 90 mL/minute, about 85 mL/minute,about 80 mL/minute, about 75 mL/minute, about 70 mL/minute, about 65mL/minute, about 60 mL/minute, about 55 mL/minute, or about 50 mL/minute(inclusive); between about 50 mL/minute to about 100 L/minute, about 95L/minute, about 90 L/minute, about 85 L/minute, about 80 L/minute, about75 L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute,about 55 L/minute, about 50 L/minute, about 45 L/minute, about 40L/minute, about 35 L/minute, about 30 L/minute, about 25 L/minute, about20 L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute,about 1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 95 mL/minute, about 90 mL/minute, about 85 mL/minute,about 80 mL/minute, about 75 mL/minute, about 70 mL/minute, about 65mL/minute, about 60 mL/minute, or about 55 mL/minute (inclusive);between about 55 mL/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute, about55 L/minute, about 50 L/minute, about 45 L/minute, about 40 L/minute,about 35 L/minute, about 30 L/minute, about 25 L/minute, about 20L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 95 mL/minute, about 90 mL/minute, about 85 mL/minute,about 80 mL/minute, about 75 mL/minute, about 70 mL/minute, about 65mL/minute, or about 60 mL/minute (inclusive); between about 60 mL/minuteto about 100 L/minute, about 95 L/minute, about 90 L/minute, about 85L/minute, about 80 L/minute, about 75 L/minute, about 70 L/minute, about65 L/minute, about 60 L/minute, about 55 L/minute, about 50 L/minute,about 45 L/minute, about 40 L/minute, about 35 L/minute, about 30L/minute, about 25 L/minute, about 20 L/minute, about 15 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 95 mL/minute, about 90 mL/minute,about 85 mL/minute, about 80 mL/minute, about 75 mL/minute, about 70mL/minute, or about 65 mL/minute (inclusive); between about 65 mL/minuteto about 100 L/minute, about 95 L/minute, about 90 L/minute, about 85L/minute, about 80 L/minute, about 75 L/minute, about 70 L/minute, about65 L/minute, about 60 L/minute, about 55 L/minute, about 50 L/minute,about 45 L/minute, about 40 L/minute, about 35 L/minute, about 30L/minute, about 25 L/minute, about 20 L/minute, about 15 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, about 100 mL/minute, about 95 mL/minute, about 90 mL/minute,about 85 mL/minute, about 80 mL/minute, about 75 mL/minute, or about 70mL/minute (inclusive); between about 70 mL/minute to about 100 L/minute,about 95 L/minute, about 90 L/minute, about 85 L/minute, about 80L/minute, about 75 L/minute, about 70 L/minute, about 65 L/minute, about60 L/minute, about 55 L/minute, about 50 L/minute, about 45 L/minute,about 40 L/minute, about 35 L/minute, about 30 L/minute, about 25L/minute, about 20 L/minute, about 15 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 95 mL/minute, about 90 mL/minute, about 85 mL/minute,about 80 mL/minute, or about 75 mL/minute (inclusive); between about 75mL/minute to about 100 L/minute, about 95 L/minute, about 90 L/minute,about 85 L/minute, about 80 L/minute, about 75 L/minute, about 70L/minute, about 65 L/minute, about 60 L/minute, about 55 L/minute, about50 L/minute, about 45 L/minute, about 40 L/minute, about 35 L/minute,about 30 L/minute, about 25 L/minute, about 20 L/minute, about 15L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about950 mL/minute, about 900 mL/minute, about 850 mL/minute, about 800mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, about 150 mL/minute, about 100 mL/minute, about 95 mL/minute,about 90 mL/minute, about 85 mL/minute, or about 80 mL/minute(inclusive); between about 80 mL/minute to about 100 L/minute, about 95L/minute, about 90 L/minute, about 85 L/minute, about 80 L/minute, about75 L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute,about 55 L/minute, about 50 L/minute, about 45 L/minute, about 40L/minute, about 35 L/minute, about 30 L/minute, about 25 L/minute, about20 L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute,about 1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 95 mL/minute, about 90 mL/minute, or about 85 mL/minute(inclusive); between about 85 mL/minute to about 100 L/minute, about 95L/minute, about 90 L/minute, about 85 L/minute, about 80 L/minute, about75 L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute,about 55 L/minute, about 50 L/minute, about 45 L/minute, about 40L/minute, about 35 L/minute, about 30 L/minute, about 25 L/minute, about20 L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute,about 1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, about 95 mL/minute, or about 90 mL/minute (inclusive);between about 90 mL/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute, about55 L/minute, about 50 L/minute, about 45 L/minute, about 40 L/minute,about 35 L/minute, about 30 L/minute, about 25 L/minute, about 20L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, about 400mL/minute, about 350 mL/minute, about 300 mL/minute, about 250mL/minute, about 200 mL/minute, about 150 mL/minute, about 100mL/minute, or about 95 mL/minute (inclusive); between about 95 mL/minuteto about 100 L/minute, about 95 L/minute, about 90 L/minute, about 85L/minute, about 80 L/minute, about 75 L/minute, about 70 L/minute, about65 L/minute, about 60 L/minute, about 55 L/minute, about 50 L/minute,about 45 L/minute, about 40 L/minute, about 35 L/minute, about 30L/minute, about 25 L/minute, about 20 L/minute, about 15 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, about 250 mL/minute, about 200 mL/minute, about 150mL/minute, or about 100 mL/minute (inclusive); between about 100mL/minute to about 100 L/minute, about 95 L/minute, about 90 L/minute,about 85 L/minute, about 80 L/minute, about 75 L/minute, about 70L/minute, about 65 L/minute, about 60 L/minute, about 55 L/minute, about50 L/minute, about 45 L/minute, about 40 L/minute, about 35 L/minute,about 30 L/minute, about 25 L/minute, about 20 L/minute, about 15L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about950 mL/minute, about 900 mL/minute, about 850 mL/minute, about 800mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, about 200mL/minute, or about 150 mL/minute (inclusive); between about 150mL/minute to about 100 L/minute, about 95 L/minute, about 90 L/minute,about 85 L/minute, about 80 L/minute, about 75 L/minute, about 70L/minute, about 65 L/minute, about 60 L/minute, about 55 L/minute, about50 L/minute, about 45 L/minute, about 40 L/minute, about 35 L/minute,about 30 L/minute, about 25 L/minute, about 20 L/minute, about 15L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about950 mL/minute, about 900 mL/minute, about 850 mL/minute, about 800mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, about 300 mL/minute, about 250 mL/minute, or about 200mL/minute (inclusive); between about 200 mL/minute to about 100L/minute, about 95 L/minute, about 90 L/minute, about 85 L/minute, about80 L/minute, about 75 L/minute, about 70 L/minute, about 65 L/minute,about 60 L/minute, about 55 L/minute, about 50 L/minute, about 45L/minute, about 40 L/minute, about 35 L/minute, about 30 L/minute, about25 L/minute, about 20 L/minute, about 15 L/minute, about 10 L/minute,about 5 L/minute, about 1 L/minute, about 950 mL/minute, about 900mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, about 350 mL/minute, about 300mL/minute, or about 250 mL/minute (inclusive); between about 250mL/minute to about 100 L/minute, about 95 L/minute, about 90 L/minute,about 85 L/minute, about 80 L/minute, about 75 L/minute, about 70L/minute, about 65 L/minute, about 60 L/minute, about 55 L/minute, about50 L/minute, about 45 L/minute, about 40 L/minute, about 35 L/minute,about 30 L/minute, about 25 L/minute, about 20 L/minute, about 15L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about950 mL/minute, about 900 mL/minute, about 850 mL/minute, about 800mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, about 550 mL/minute, about 500mL/minute, about 450 mL/minute, about 400 mL/minute, about 350mL/minute, or about 300 mL/minute (inclusive); about 300 mL/minute toabout 100 L/minute, about 95 L/minute, about 90 L/minute, about 85L/minute, about 80 L/minute, about 75 L/minute, about 70 L/minute, about65 L/minute, about 60 L/minute, about 55 L/minute, about 50 L/minute,about 45 L/minute, about 40 L/minute, about 35 L/minute, about 30L/minute, about 25 L/minute, about 20 L/minute, about 15 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 950 mL/minute,about 900 mL/minute, about 850 mL/minute, about 800 mL/minute, about 750mL/minute, about 700 mL/minute, about 650 mL/minute, about 600mL/minute, about 550 mL/minute, about 500 mL/minute, about 450mL/minute, about 400 mL/minute, or about 350 mL/minute (inclusive);between about 350 mL/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute, about55 L/minute, about 50 L/minute, about 45 L/minute, about 40 L/minute,about 35 L/minute, about 30 L/minute, about 25 L/minute, about 20L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, about 450 mL/minute, or about 400mL/minute (inclusive); about 400 mL/minute to about 100 L/minute, about95 L/minute, about 90 L/minute, about 85 L/minute, about 80 L/minute,about 75 L/minute, about 70 L/minute, about 65 L/minute, about 60L/minute, about 55 L/minute, about 50 L/minute, about 45 L/minute, about40 L/minute, about 35 L/minute, about 30 L/minute, about 25 L/minute,about 20 L/minute, about 15 L/minute, about 10 L/minute, about 5L/minute, about 1 L/minute, about 950 mL/minute, about 900 mL/minute,about 850 mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, about 500 mL/minute, or about 450 mL/minute (inclusive);between about 450 mL/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute, about55 L/minute, about 50 L/minute, about 45 L/minute, about 40 L/minute,about 35 L/minute, about 30 L/minute, about 25 L/minute, about 20L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, about 600 mL/minute, about 550mL/minute, or about 500 mL/minute (inclusive); between about 500mL/minute to about 100 L/minute, about 95 L/minute, about 90 L/minute,about 85 L/minute, about 80 L/minute, about 75 L/minute, about 70L/minute, about 65 L/minute, about 60 L/minute, about 55 L/minute, about50 L/minute, about 45 L/minute, about 40 L/minute, about 35 L/minute,about 30 L/minute, about 25 L/minute, about 20 L/minute, about 15L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about950 mL/minute, about 900 mL/minute, about 850 mL/minute, about 800mL/minute, about 750 mL/minute, about 700 mL/minute, about 650mL/minute, about 600 mL/minute, or about 550 mL/minute (inclusive);between about 550 mL/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute, about55 L/minute, about 50 L/minute, about 45 L/minute, about 40 L/minute,about 35 L/minute, about 30 L/minute, about 25 L/minute, about 20L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, about 650 mL/minute, or about 600 mL/minute (inclusive);between about 600 mL/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute, about55 L/minute, about 50 L/minute, about 45 L/minute, about 40 L/minute,about 35 L/minute, about 30 L/minute, about 25 L/minute, about 20L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, about 750 mL/minute, about 700mL/minute, or about 650 mL/minute (inclusive); between about 650mL/minute to about 100 L/minute, about 95 L/minute, about 90 L/minute,about 85 L/minute, about 80 L/minute, about 75 L/minute, about 70L/minute, about 65 L/minute, about 60 L/minute, about 55 L/minute, about50 L/minute, about 45 L/minute, about 40 L/minute, about 35 L/minute,about 30 L/minute, about 25 L/minute, about 20 L/minute, about 15L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about950 mL/minute, about 900 mL/minute, about 850 mL/minute, about 800mL/minute, about 750 mL/minute, or about 700 mL/minute (inclusive);between about 700 mL/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute, about55 L/minute, about 50 L/minute, about 45 L/minute, about 40 L/minute,about 35 L/minute, about 30 L/minute, about 25 L/minute, about 20L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, about 800 mL/minute, or about 750 mL/minute (inclusive);between about 750 mL/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute, about55 L/minute, about 50 L/minute, about 45 L/minute, about 40 L/minute,about 35 L/minute, about 30 L/minute, about 25 L/minute, about 20L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, about 900 mL/minute, about 850mL/minute, or about 800 mL/minute (inclusive); between about 800mL/minute to about 100 L/minute, about 95 L/minute, about 90 L/minute,about 85 L/minute, about 80 L/minute, about 75 L/minute, about 70L/minute, about 65 L/minute, about 60 L/minute, about 55 L/minute, about50 L/minute, about 45 L/minute, about 40 L/minute, about 35 L/minute,about 30 L/minute, about 25 L/minute, about 20 L/minute, about 15L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about950 mL/minute, about 900 mL/minute, or about 850 mL/minute (inclusive);between about 850 mL/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute, about55 L/minute, about 50 L/minute, about 45 L/minute, about 40 L/minute,about 35 L/minute, about 30 L/minute, about 25 L/minute, about 20L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 950 mL/minute, or about 900 mL/minute (inclusive);between about 900 mL/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute, about55 L/minute, about 50 L/minute, about 45 L/minute, about 40 L/minute,about 35 L/minute, about 30 L/minute, about 25 L/minute, about 20L/minute, about 15 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, or about 950 mL/minute (inclusive); between about 950mL/minute to about 100 L/minute, about 95 L/minute, about 90 L/minute,about 85 L/minute, about 80 L/minute, about 75 L/minute, about 70L/minute, about 65 L/minute, about 60 L/minute, about 55 L/minute, about50 L/minute, about 45 L/minute, about 40 L/minute, about 35 L/minute,about 30 L/minute, about 25 L/minute, about 20 L/minute, about 15L/minute, about 10 L/minute, about 5 L/minute, or about 1 L/minute(inclusive); between about 1 L/minute to about 100 L/minute, about 95L/minute, about 90 L/minute, about 85 L/minute, about 80 L/minute, about75 L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute,about 55 L/minute, about 50 L/minute, about 45 L/minute, about 40L/minute, about 35 L/minute, about 30 L/minute, about 25 L/minute, about20 L/minute, about 15 L/minute, about 10 L/minute, or about 5 L/minute(inclusive); between about 5 L/minute to about 100 L/minute, about 95L/minute, about 90 L/minute, about 85 L/minute, about 80 L/minute, about75 L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute,about 55 L/minute, about 50 L/minute, about 45 L/minute, about 40L/minute, about 35 L/minute, about 30 L/minute, about 25 L/minute, about20 L/minute, about 15 L/minute, or about 10 L/minute (inclusive);between about 10 L/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute, about55 L/minute, about 50 L/minute, about 45 L/minute, about 40 L/minute,about 35 L/minute, about 30 L/minute, about 25 L/minute, about 20L/minute, or about 15 L/minute (inclusive); between about 15 L/minute toabout 100 L/minute, about 95 L/minute, about 90 L/minute, about 85L/minute, about 80 L/minute, about 75 L/minute, about 70 L/minute, about65 L/minute, about 60 L/minute, about 55 L/minute, about 50 L/minute,about 45 L/minute, about 40 L/minute, about 35 L/minute, about 30L/minute, about 25 L/minute, or about 20 L/minute (inclusive); betweenabout 20 L/minute to about 100 L/minute, about 95 L/minute, about 90L/minute, about 85 L/minute, about 80 L/minute, about 75 L/minute, about70 L/minute, about 65 L/minute, about 60 L/minute, about 55 L/minute,about 50 L/minute, about 45 L/minute, about 40 L/minute, about 35L/minute, about 30 L/minute, or about 25 L/minute (inclusive); betweenabout 25 L/minute to about 100 L/minute, about 95 L/minute, about 90L/minute, about 85 L/minute, about 80 L/minute, about 75 L/minute, about70 L/minute, about 65 L/minute, about 60 L/minute, about 55 L/minute,about 50 L/minute, about 45 L/minute, about 40 L/minute, about 35L/minute, or about 30 L/minute (inclusive); between about 30 L/minute toabout 100 L/minute, about 95 L/minute, about 90 L/minute, about 85L/minute, about 80 L/minute, about 75 L/minute, about 70 L/minute, about65 L/minute, about 60 L/minute, about 55 L/minute, about 50 L/minute,about 45 L/minute, about 40 L/minute, or about 35 L/minute (inclusive);between about 35 L/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute, about55 L/minute, about 50 L/minute, about 45 L/minute, or about 40 L/minute(inclusive); between about 40 L/minute to about 100 L/minute, about 95L/minute, about 90 L/minute, about 85 L/minute, about 80 L/minute, about75 L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute,about 55 L/minute, about 50 L/minute, or about 45 L/minute (inclusive);between about 45 L/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, about 70 L/minute, about 65 L/minute, about 60 L/minute, about55 L/minute, or about 50 L/minute (inclusive); between about 50 L/minuteto about 100 L/minute, about 95 L/minute, about 90 L/minute, about 85L/minute, about 80 L/minute, about 75 L/minute, about 70 L/minute, about65 L/minute, about 60 L/minute, or about 55 L/minute (inclusive);between about 55 L/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, about 70 L/minute, about 65 L/minute, or about 60 L/minute(inclusive); between about 60 L/minute to about 100 L/minute, about 95L/minute, about 90 L/minute, about 85 L/minute, about 80 L/minute, about75 L/minute, about 70 L/minute, or about 65 L/minute (inclusive);between about 65 L/minute to about 100 L/minute, about 95 L/minute,about 90 L/minute, about 85 L/minute, about 80 L/minute, about 75L/minute, or about 70 L/minute (inclusive); between about 70 L/minute toabout 100 L/minute, about 95 L/minute, about 90 L/minute, about 85L/minute, about 80 L/minute, or about 75 L/minute (inclusive); betweenabout 75 L/minute to about 100 L/minute, about 95 L/minute, about 90L/minute, about 85 L/minute, or about 80 L/minute (inclusive); betweenabout 80 L/minute to about 100 L/minute, about 95 L/minute, about 90L/minute, or about 85 L/minute (inclusive); between about 85 L/minute toabout 100 L/minute, about 95 L/minute, or about 90 L/minute (inclusive);between about 90 L/minute to about 100 L/minute or about 95 L/minute(inclusive); or about 95 L/minute to about 100 L/minute (inclusive). Theliquid culture medium containing the recombinant therapeutic protein canbe derived from any of the exemplary sources described herein or knownin the art.

Some examples further include the optional step of filtering the liquidculture medium before it is fed onto the first MCCS. Any of theexemplary means of filtering a liquid culture medium or a fluidcontaining the recombinant therapeutic protein described herein, or anyfiltration means known in the art, can be used to filter the liquidculture medium containing the recombinant therapeutic protein before itis fed into the first MCCS.

In the methods described herein, the capturing of the recombinanttherapeutic protein from the liquid culture medium is performed using afirst MCCS. As can be appreciated in the art, in order to achieve thecapture of the recombinant therapeutic protein, at least onechromatographic column or at least one chromatographic membrane in thefirst MCCS must contain a resin that utilizes a capturing mechanism(e.g., any of the exemplary capturing mechanisms described herein), orcontains a resin capable of performing cation exchange, anion exchange,or molecule sieve chromatography. For example, if the recombinanttherapeutic protein is an antibody or an antibody fragment, thecapturing system can be a protein A-binding capturing mechanism or anantigen-binding capturing mechanism (where the capturing antigen isspecifically recognized by the recombinant therapeutic antibody orantibody fragment). If the recombinant therapeutic protein is an enzyme,the capturing mechanism can use an antibody or antibody fragment thatspecifically binds to the enzyme to capture the recombinant therapeuticenzyme, a substrate of the enzyme to capture the recombinant therapeuticenzyme, a cofactor of the enzyme to capture the recombinant therapeuticenzyme, or, if the recombinant therapeutic enzyme contains a tag, aprotein, metal chelate, or antibody (or antibody fragment) thatspecifically binds to the tag present in the recombinant therapeuticenzyme. Non-limiting resins that can be used to capture a recombinanttherapeutic protein are described herein and additional resins that canbe used to capture a recombinant therapeutic protein are known in theart. One non-limiting example of resin that utilizes a protein A-bindingcapture mechanism is MabSelect SuRe resin (GE Healthcare, Piscataway,N.J.).

Exemplary non-limiting sizes and shapes of the chromatography column(s)or chromatographic membrane(s) present in the first MCCS that can beused to capture the recombinant therapeutic protein are describedherein. The liquid culture medium fed (loaded) into the first MCCS cancontain, e.g., between about 0.05 mg/mL to about 100 mg/mL recombinanttherapeutic protein (e.g., between about 0.1 mg/mL to about 90 mg/mL,between about 0.1 mg/mL to about 80 mg/mL, between about 0.1 mg/mL toabout 70 mg/mL, between about 0.1 mg/mL to about 60 mg/mL, between about0.1 mg/mL to about 50 mg/mL, between about 0.1 mg/mL to about 40 mg/mL,between about 0.1 mg/mL to about 30 mg/mL, between about 0.1 mg/mL toabout 20 mg/mL, between 0.5 mg/mL to about 20 mg/mL, between about 0.1mg/mL to about 15 mg/mL, between about 0.5 mg/mL to about 15 mg/mL,between about 0.1 mg/mL to about 10 mg/mL, or between about 0.5 mg/mL toabout 10 mg/mL recombinant therapeutic protein). The mean time requiredfor the recombinant therapeutic protein to bind to the resin used toperform the unit operation of capturing can be, e.g., between about 1second to about 24 hours, about 20 hours, about 16 hours, about 12hours, about 8 hours, about 4 hours, about 3 hours, about 2 hours, about1 hour, about 55 minutes, about 50 minutes, about 45 minutes, about 40minutes, about 35 minutes, about 30 minutes, about 25 minutes, about 20minutes, about 18 minutes, about 16 minutes, about 14 minutes, about 12minutes, about 10 minutes, about 8 minutes, about 6 minutes, about 5minutes, about 4 minutes, about 3 minutes, about 2 minutes, about 1minute, about 30 seconds, about 20 seconds, about 10 seconds, or about 5seconds (inclusive); between about 5 seconds to about 24 hours, about 20hours, about 16 hours, about 12 hours, about 8 hours, about 4 hours,about 3 hours, about 2 hours, about 1 hour, about 55 minutes, about 50minutes, about 45 minutes, about 40 minutes, about 35 minutes, about 30minutes, about 25 minutes, about 20 minutes, about 18 minutes, about 16minutes, about 14 minutes, about 12 minutes, about 10 minutes, about 8minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3minutes, about 2 minutes, about 1 minute, about 30 seconds, about 20seconds, or about 10 seconds (inclusive); between about 10 seconds toabout 24 hours, about 20 hours, about 16 hours, about 12 hours, about 8hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, about35 minutes, about 30 minutes, about 25 minutes, about 20 minutes, about18 minutes, about 16 minutes, about 14 minutes, about 12 minutes, about10 minutes, about 8 minutes, about 6 minutes, about 5 minutes, about 4minutes, about 3 minutes, about 2 minutes, about 1 minute, about 30seconds, or about 20 seconds (inclusive); between about 20 seconds toabout 24 hours, about 20 hours, about 16 hours, about 12 hours, about 8hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, about35 minutes, about 30 minutes, about 25 minutes, about 20 minutes, about18 minutes, about 16 minutes, about 14 minutes, about 12 minutes, about10 minutes, about 8 minutes, about 6 minutes, about 5 minutes, about 4minutes, about 3 minutes, about 2 minutes, about 1 minute, or about 30seconds (inclusive); between about 30 seconds to about 24 hours, about20 hours, about 16 hours, about 12 hours, about 8 hours, about 4 hours,about 3 hours, about 2 hours, about 1 hour, about 55 minutes, about 50minutes, about 45 minutes, about 40 minutes, about 35 minutes, about 30minutes, about 25 minutes, about 20 minutes, about 18 minutes, about 16minutes, about 14 minutes, about 12 minutes, about 10 minutes, about 8minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3minutes, about 2 minutes, or about 1 minute (inclusive); between about 1minute to about 24 hours, about 20 hours, about 16 hours, about 12hours, about 8 hours, about 4 hours, about 3 hours, about 2 hours, about1 hour, about 55 minutes, about 50 minutes, about 45 minutes, about 40minutes, about 35 minutes, about 30 minutes, about 25 minutes, about 20minutes, about 18 minutes, about 16 minutes, about 14 minutes, about 12minutes, about 10 minutes, about 8 minutes, about 6 minutes, about 5minutes, about 4 minutes, about 3 minutes, or about 2 minutes(inclusive); between about 2 minutes to about 24 hours, about 20 hours,about 16 hours, about 12 hours, about 8 hours, about 4 hours, about 3hours, about 2 hours, about 1 hour, about 55 minutes, about 50 minutes,about 45 minutes, about 40 minutes, about 35 minutes, about 30 minutes,about 25 minutes, about 20 minutes, about 18 minutes, about 16 minutes,about 14 minutes, about 12 minutes, about 10 minutes, about 8 minutes,about 6 minutes, about 5 minutes, about 4 minutes, or about 3 minutes(inclusive); between about 3 minutes to about 24 hours, about 20 hours,about 16 hours, about 12 hours, about 8 hours, about 4 hours, about 3hours, about 2 hours, about 1 hour, about 55 minutes, about 50 minutes,about 45 minutes, about 40 minutes, about 35 minutes, about 30 minutes,about 25 minutes, about 20 minutes, about 18 minutes, about 16 minutes,about 14 minutes, about 12 minutes, about 10 minutes, about 8 minutes,about 6 minutes, about 5 minutes, or about 4 minutes (inclusive);between about 4 minutes to about 24 hours, about 20 hours, about 16hours, about 12 hours, about 8 hours, about 4 hours, about 3 hours,about 2 hours, about 1 hour, about 55 minutes, about 50 minutes, about45 minutes, about 40 minutes, about 35 minutes, about 30 minutes, about25 minutes, about 20 minutes, about 18 minutes, about 16 minutes, about14 minutes, about 12 minutes, about 10 minutes, about 8 minutes, about 6minutes, or about 5 minutes (inclusive); between about 5 minutes toabout 24 hours, about 20 hours, about 16 hours, about 12 hours, about 8hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, about35 minutes, about 30 minutes, about 25 minutes, about 20 minutes, about18 minutes, about 16 minutes, about 14 minutes, about 12 minutes, about10 minutes, about 8 minutes, or about 6 minutes (inclusive); betweenabout 6 minutes to about 24 hours, about 20 hours, about 16 hours, about12 hours, about 8 hours, about 4 hours, about 3 hours, about 2 hours,about 1 hour, about 55 minutes, about 50 minutes, about 45 minutes,about 40 minutes, about 35 minutes, about 30 minutes, about 25 minutes,about 20 minutes, about 18 minutes, about 16 minutes, about 14 minutes,about 12 minutes, about 10 minutes, or about 8 minutes (inclusive);between about 8 minutes to about 60 minutes, about 55 minutes, about 50minutes, about 45 minutes, about 40 minutes, about 35 minutes, about 30minutes, about 25 minutes, about 20 minutes, about 18 minutes, about 16minutes, about 14 minutes, about 12 minutes, or about 10 minutes(inclusive); between about 10 minutes to about 24 hours, about 20 hours,about 16 hours, about 12 hours, about 8 hours, about 4 hours, about 3hours, about 2 hours, about 1 hour, about 55 minutes, about 50 minutes,about 45 minutes, about 40 minutes, about 35 minutes, about 30 minutes,about 25 minutes, about 20 minutes, about 18 minutes, about 16 minutes,about 14 minutes, or about 12 minutes (inclusive); between about 12minutes to about 24 hours, about 20 hours, about 16 hours, about 12hours, about 8 hours, about 4 hours, about 3 hours, about 2 hours, about1 hour, about 55 minutes, about 50 minutes, about 45 minutes, about 40minutes, about 35 minutes, about 30 minutes, about 25 minutes, about 20minutes, about 18 minutes, about 16 minutes, or about 14 minutes(inclusive); between about 14 minutes to about 24 hours, about 20 hours,about 16 hours, about 12 hours, about 8 hours, about 4 hours, about 3hours, about 2 hours, about 1 hour, about 55 minutes, about 50 minutes,about 45 minutes, about 40 minutes, about 35 minutes, about 30 minutes,about 25 minutes, about 20 minutes, about 18 minutes, or about 16minutes (inclusive); between about 16 minutes to about 60 minutes, about55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, about35 minutes, about 30 minutes, about 25 minutes, about 20 minutes, orabout 18 minutes (inclusive); between about 18 minutes to about 24hours, about 20 hours, about 16 hours, about 12 hours, about 8 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 55minutes, about 50 minutes, about 45 minutes, about 40 minutes, about 35minutes, about 30 minutes, about 25 minutes, or about 20 minutes(inclusive); between about 20 minutes to about 24 hours, about 20 hours,about 16 hours, about 12 hours, about 8 hours, about 4 hours, about 3hours, about 2 hours, about 1 hour, about 55 minutes, about 50 minutes,about 45 minutes, about 40 minutes, about 35 minutes, about 30 minutes,or about 25 minutes (inclusive); between about 25 minutes to about 24hours, about 20 hours, about 16 hours, about 12 hours, about 8 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 55minutes, about 50 minutes, about 45 minutes, about 40 minutes, about 35minutes, or about 30 minutes (inclusive); between about 30 minutes toabout 24 hours, about 20 hours, about 16 hours, about 12 hours, about 8hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, orabout 35 minutes (inclusive); between about 35 minutes to about 24hours, about 20 hours, about 16 hours, about 12 hours, about 8 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 55minutes, about 50 minutes, about 45 minutes, or about 40 minutes(inclusive); between about 40 minutes to about 24 hours, about 20 hours,about 16 hours, about 12 hours, about 8 hours, about 4 hours, about 3hours, about 2 hours, about 1 hour, about 55 minutes, about 50 minutes,or about 45 minutes (inclusive); between about 45 minutes to about 24hours, about 20 hours, about 16 hours, about 12 hours, about 8 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 55minutes, or about 50 minutes (inclusive); between about 50 minutes toabout 24 hours, about 20 hours, about 16 hours, about 12 hours, about 8hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, orabout 55 minutes (inclusive); about 55 minutes to about 24 hours, about20 hours, about 16 hours, about 12 hours, about 8 hours, about 4 hours,about 3 hours, about 2 hours, about 1 hour, or about 60 minutes(inclusive); between about 60 minutes to about 24 hours, about 20 hours,about 16 hours, about 12 hours, about 8 hours, about 4 hours, about 3hours, about 2 hours, or about 1 hour (inclusive); between about 1 hourto about 24 hours, about 20 hours, about 16 hours, about 12 hours, about8 hours, about 4 hours, about 3 hours, or about 2 hours (inclusive);between about 2 hours to about 24 hours, about 20 hours, about 16 hours,about 12 hours, about 8 hours, about 4 hours, or about 3 hours(inclusive); between about 3 hours to about 24 hours, about 20 hours,about 16 hours, about 12 hours, about 8 hours, or about 4 hours(inclusive); between about 4 hours to about 24 hours, about 20 hours,about 16 hours, about 12 hours, or about 8 hours (inclusive); betweenabout 8 hours to about 24 hours, about 20 hours, about 16 hours, orabout 12 hours (inclusive); between about 12 hours to about 24 hours,about 20 hours, or about 16 hours (inclusive); between about 16 hours toabout 24 hours or about 20 hours (inclusive); or between about 20 hoursto about 24 hours (inclusive).

As can be appreciated in the art, in order to capture the recombinanttherapeutic protein using the chromatography column(s) orchromatographic membrane(s) present in the first MCCS, one must performthe sequential chromatographic steps of loading, washing, eluting, andregenerating the chromatography column(s) or chromatography membrane(s)present in the first MCCS. Any of the exemplary flow rates, buffervolumes, and/or lengths of time allotted for each sequentialchromatographic step described herein can be used in the one or more ofthese different sequential chromatographic steps (e.g., one or more ofthe sequential chromatographic steps of loading, washing, eluting, andregenerating the chromatography column(s) or chromatography membrane(s)present in the first MCCS that are used for capturing the recombinanttherapeutic protein). Non-limiting flow rates, buffer volumes, and/orlengths of time allotted for each sequential chromatographic step thatcan be used for capturing chromatographic column(s) and/orchromatographic membrane(s) in the first MCCS (e.g., a first PCC system)are provided below. In addition, exemplary buffers elution buffers thatcan be used in the first MCCS are described below.

The first MCCS containing at least one chromatographic column and/orchromatographic membrane containing a resin that can perform the unitoperation of capturing (e.g., any of exemplary resins that can be usedfor capturing described herein) can be loaded with the liquid culturemedium containing a recombinant therapeutic protein using any of loadingflow rates (fed rates) described above. In some examples, a singlechromatographic column or single chromatographic membrane containing aresin that is capable of performing the unit operation of capturing isloaded in, e.g., between about 10 minutes to about 48 hours, about 44hours, about 40 hours, about 36 hours, about 32 hours, about 28 hours,about 24 hours, about 20 hours, about 16 hours, about 12 hours, about 8hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about3 hours, about 2 hours, about 110 minutes, about 100 minutes, about 90minutes, about 80 minutes, about 70 minutes, about 60 minutes, about 50minutes, about 40 minutes, about 30 minutes, about 20 minutes, or about15 minutes (inclusive); between about 15 minutes to about 48 hours,about 44 hours, about 40 hours, about 36 hours, about 32 hours, about 28hours, about 24 hours, about 20 hours, about 16 hours, about 12 hours,about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4hours, about 3 hours, about 2 hours, about 110 minutes, about 100minutes, about 90 minutes, about 80 minutes, about 70 minutes, about 60minutes, about 50 minutes, about 40 minutes, about 30 minutes, or about20 minutes (inclusive); between about 20 minutes to about 48 hours,about 44 hours, about 40 hours, about 36 hours, about 32 hours, about 28hours, about 24 hours, about 20 hours, about 16 hours, about 12 hours,about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4hours, about 3 hours, about 2 hours, about 110 minutes, about 100minutes, about 90 minutes, about 80 minutes, about 70 minutes, about 60minutes, about 50 minutes, about 40 minutes, or about 30 minutes(inclusive); between about 30 minutes to about 48 hours, about 44 hours,about 40 hours, about 36 hours, about 32 hours, about 28 hours, about 24hours, about 20 hours, about 16 hours, about 12 hours, about 8 hours,about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3hours, about 2 hours, about 110 minutes, about 100 minutes, about 90minutes, about 80 minutes, about 70 minutes, about 60 minutes, about 50minutes, or about 40 minutes (inclusive); between about 40 minutes toabout 48 hours, about 44 hours, about 40 hours, about 36 hours, about 32hours, about 28 hours, about 24 hours, about 20 hours, about 16 hours,about 12 hours, about 8 hours, about 7 hours, about 6 hours, about 5hours, about 4 hours, about 3 hours, about 2 hours, about 110 minutes,about 100 minutes, about 90 minutes, about 80 minutes, about 70 minutes,about 60 minutes, or about 50 minutes (inclusive); between about 50minutes to about 48 hours, about 44 hours, about 40 hours, about 36hours, about 32 hours, about 28 hours, about 24 hours, about 20 hours,about 16 hours, about 12 hours, about 8 hours, about 7 hours, about 6hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about110 minutes, about 100 minutes, about 90 minutes, about 80 minutes,about 70 minutes, or about 60 minutes (inclusive); between about 60minutes to about 48 hours, about 44 hours, about 40 hours, about 36hours, about 32 hours, about 28 hours, about 24 hours, about 20 hours,about 16 hours, about 12 hours, about 8 hours, about 7 hours, about 6hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about110 minutes, about 100 minutes, about 90 minutes, about 80 minutes, orabout 70 minutes (inclusive); between about 70 minutes to about 48hours, about 44 hours, about 40 hours, about 36 hours, about 32 hours,about 28 hours, about 24 hours, about 20 hours, about 16 hours, about 12hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about4 hours, about 3 hours, about 2 hours, about 110 minutes, about 100minutes, about 90 minutes, or about 80 minutes (inclusive); betweenabout 80 minutes to about 48 hours, about 44 hours, about 40 hours,about 36 hours, about 32 hours, about 28 hours, about 24 hours, about 20hours, about 16 hours, about 12 hours, about 8 hours, about 7 hours,about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2hours, about 110 minutes, about 100 minutes, or about 90 minutes(inclusive); between about 90 minutes to about 48 hours, about 44 hours,about 40 hours, about 36 hours, about 32 hours, about 28 hours, about 24hours, about 20 hours, about 16 hours, about 12 hours, about 8 hours,about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3hours, about 2 hours, about 110 minutes, or about 100 minutes(inclusive); between about 100 minutes to about 48 hours, about 44hours, about 40 hours, about 36 hours, about 32 hours, about 28 hours,about 24 hours, about 20 hours, about 16 hours, about 12 hours, about 8hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about3 hours, about 2 hours, or about 110 minutes (inclusive); between about110 minutes to about 48 hours, about 44 hours, about 40 hours, about 36hours, about 32 hours, about 28 hours, about 24 hours, about 20 hours,about 16 hours, about 12 hours, about 8 hours, about 7 hours, about 6hours, about 5 hours, about 4 hours, about 3 hours, or about 2 hours(inclusive); between about 2 hours to about 48 hours, about 44 hours,about 40 hours, about 36 hours, about 32 hours, about 28 hours, about 24hours, about 20 hours, about 16 hours, about 12 hours, about 8 hours,about 7 hours, about 6 hours, about 5 hours, about 4 hours, or about 3hours (inclusive); between about 3 hours to about 48 hours, about 44hours, about 40 hours, about 36 hours, about 32 hours, about 28 hours,about 24 hours, about 20 hours, about 16 hours, about 12 hours, about 8hours, about 7 hours, about 6 hours, about 5 hours, or about 4 hours(inclusive); between about 4 hours to about 48 hours, about 44 hours,about 40 hours, about 36 hours, about 32 hours, about 28 hours, about 24hours, about 20 hours, about 16 hours, about 12 hours, about 8 hours,about 7 hours, about 6 hours, or about 5 hours (inclusive); betweenabout 5 hours to about 48 hours, about 44 hours, about 40 hours, about36 hours, about 32 hours, about 28 hours, about 24 hours, about 20hours, about 16 hours, about 12 hours, about 8 hours, about 7 hours, orabout 6 hours (inclusive); between about 6 hours to about 48 hours,about 44 hours, about 40 hours, about 36 hours, about 32 hours, about 28hours, about 24 hours, about 20 hours, about 16 hours, about 12 hours,about 8 hours, or about 7 hours (inclusive); between about 7 hours toabout 48 hours, about 44 hours, about 40 hours, about 36 hours, about 32hours, about 28 hours, about 24 hours, about 20 hours, about 16 hours,about 12 hours, or about 8 hours (inclusive); between about 8 hours toabout 48 hours, about 44 hours, about 40 hours, about 36 hours, about 32hours, about 28 hours, about 24 hours, about 20 hours, about 16 hours,or about 12 hours (inclusive); between about 12 hours to about 48 hours,about 44 hours, about 40 hours, about 36 hours, about 32 hours, about 28hours, about 24 hours, about 20 hours, or about 16 hours (inclusive);between about 16 hours to about 48 hours, about 44 hours, about 40hours, about 36 hours, about 32 hours, about 28 hours, about 24 hours,or about 20 hours (inclusive); between about 20 hours to about 48 hours,about 44 hours, about 40 hours, about 36 hours, about 32 hours, about 28hours, or about 24 hours (inclusive); between about 24 hours to about 48hours, about 44 hours, about 40 hours, about 36 hours, about 32 hours,or about 28 hours (inclusive); between about 28 hours to about 48 hours,about 44 hours, about 40 hours, about 36 hours, or about 32 hours(inclusive); between about 32 hours to about 48 hours, about 44 hours,about 40 hours, or about 36 hours (inclusive); between about 36 hours toabout 48 hours, about 44 hours, or about 40 hours (inclusive); betweenabout 40 hours to about 48 hours or about 44 hours (inclusive), or about44 hours to about 48 hours (inclusive). In some examples, wherein thefirst MCCS includes at least two chromatographic columns that contain aresin that is capable of performing the unit operation of capturing inseries, the time required to load two of the chromatographic columns inseries is, e.g., between about 50 minutes to about 180 minutes (e.g.,between about 60 minutes and about 180 minutes, between about 70 minutesand about 180 minutes, between about 80 minutes and about 180 minutes,between about 90 minutes and about 180 minutes, between about 100minutes and about 180 minutes, between about 110 minutes and 150minutes, and between about 125 minutes and about 145 minutes).

Following the loading of the recombinant therapeutic protein onto the atleast one chromatographic column or chromatographic membrane in thefirst MCCS that contains a resin that is capable of performing the unitoperation of capturing, the at least one chromatographic column orchromatographic membrane is washed with at least one washing buffer. Ascan be appreciated in the art, the at least one (e.g., two, three, orfour) washing buffer is meant to elute all proteins that are not therecombinant therapeutic protein from the at least one chromatographycolumn or chromatographic membrane, while not disturbing the interactionof the recombinant therapeutic protein with the resin.

The wash buffer can be passed through the at least one chromatographycolumn or chromatographic membrane at a flow rate of, e.g., betweenabout 0.2 mL/minute to about 100 L/minute (inclusive) (e.g., any of thesubranges of this flow rate range described herein). The volume of washbuffer used (e.g., combined total volume of wash buffer used when morethan one wash buffer is used) can be, e.g., between about 1× columnvolume (CV) to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about22×CV, about 20×CV, about 18×CV, about 16×CV, about 14×CV, about 13×CV,about 12×CV, about 11×CV, about 10×CV, about 9×CV, about 8×CV, about7×CV, about 6×CV, about 5×CV, about 4×CV, or about 3×CV (inclusive);between about 2×CV to about 30×CV, about 28×CV, about 26×CV, about24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV, about 14×CV,about 13×CV, about 12×CV, about 11×CV, about 10×CV, about 9×CV, about8×CV, about 7×CV, about 6×CV, about 5×CV, or about 4×CV (inclusive);between about 3×CV to about 30×CV, about 28×CV, about 26×CV, about24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV, about 14×CV,about 13×CV, about 12×CV, about 11×CV, about 10×CV, about 9×CV, about8×CV, about 7×CV, or about 5×CV (inclusive); between about 4×CV to about30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV,about 18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV, about11×CV, about 10×CV, about 9×CV, about 8×CV, about 7×CV, or about 6×CV(inclusive); between about 5×CV to about 30×CV, about 28×CV, about26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV,about 14×CV, about 13×CV, about 12×CV, about 11×CV, about 10×CV, about9×CV, about 8×CV, or about 7×CV (inclusive); between about 6×CV to about30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV,about 18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV, about11×CV, about 10×CV, about 9×CV, or about 8×CV (inclusive); between about7×CV to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV,about 20×CV, about 18×CV, about 16×CV, about 14×CV, about 13×CV, about12×CV, about 11×CV, about 10×CV, or about 9×CV (inclusive); betweenabout 8×CV to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about22×CV, about 20×CV, about 18×CV, about 16×CV, about 14×CV, about 13×CV,about 12×CV, about 11×CV, or about 10×CV (inclusive); between about 9 CVto about 30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV,about 20×CV, about 18×CV, about 16×CV, about 14×CV, about 13×CV, about12×CV, or about 11×CV (inclusive); between about 10×CV to about 30×CV,about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV, about18×CV, about 16×CV, about 14×CV, about 13×CV, or about 12×CV(inclusive); between about 11×CV to about 30×CV, about 28×CV, about26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV,about 14×CV, or about 13×CV (inclusive); between about 12×CV to about30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV,about 18×CV, about 16×CV, or about 14×CV (inclusive); between about14×CV to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about22×CV, about 20×CV, about 18×CV, or about 16×CV (inclusive); betweenabout 16×CV to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about22×CV, about 20×CV, or about 18×CV (inclusive); between about 18×CV toabout 30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, orabout 20×CV (inclusive); between about 20×CV to about 30×CV, about28×CV, about 26×CV, about 24×CV, or about 22×CV (inclusive); betweenabout 22×CV to about 30×CV, about 28×CV, about 26×CV, or about 24×CV(inclusive); between about 24×CV to about 30×CV, about 28×CV, or about26×CV (inclusive); between about 26×CV to about 30×CV or about 28×CV(inclusive); or between about 28×CV to about 30×CV (inclusive).

The total time of the washing can be, e.g., between about 2 minutes toabout 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16hours, about 14 hours, about 12 hours, about 10 hours, about 9 hours,about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4.5hours, about 4 hours, about 3.5 hours, about 3 hours, about 2.5 hours,about 2 hours, about 1.5 hours, about 1 hour, about 50 minutes, about 40minutes, about 30 minutes, about 20 minutes, about 10 minutes, or about5 minutes (inclusive); between about 5 minutes to about 24 hours, about22 hours, about 20 hours, about 18 hours, about 16 hours, about 14hours, about 12 hours, about 10 hours, about 9 hours, about 8 hours,about 7 hours, about 6 hours, about 5 hours, about 4.5 hours, about 4hours, about 3.5 hours, about 3 hours, about 2.5 hours, about 2 hours,about 1.5 hours, about 1 hour, about 50 minutes, about 40 minutes, about30 minutes, about 20 minutes, or about 10 minutes (inclusive); about 10minutes to about 24 hours, about 22 hours, about 20 hours, about 18hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours,about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5hours, about 4.5 hours, about 4 hours, about 3.5 hours, about 3 hours,about 2.5 hours, about 2 hours, about 1.5 hours, about 1 hour, about 50minutes, about 40 minutes, about 30 minutes, or about 20 minutes(inclusive); between about 20 minutes to about 24 hours, about 22 hours,about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours,about 6 hours, about 5 hours, about 4.5 hours, about 4 hours, about 3.5hours, about 3 hours, about 2.5 hours, about 2 hours, about 1.5 hours,about 1 hour, about 50 minutes, about 40 minutes, or about 30 minutes(inclusive); between about 30 minutes to about 24 hours, about 22 hours,about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours,about 6 hours, about 5 hours, about 4.5 hours, about 4 hours, about 3.5hours, about 3 hours, about 2.5 hours, about 2 hours, about 1.5 hours,about 1 hour, about 50 minutes, or about 40 minutes (inclusive); betweenabout 40 minutes to about 24 hours, about 22 hours, about 20 hours,about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about5 hours, about 4.5 hours, about 4 hours, about 3.5 hours, about 3 hours,about 2.5 hours, about 2 hours, about 1.5 hours, about 1 hour, or about50 minutes (inclusive); between about 50 minutes to about 24 hours,about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14hours, about 12 hours, about 10 hours, about 9 hours, about 8 hours,about 7 hours, about 6 hours, about 5 hours, about 4.5 hours, about 4hours, about 3.5 hours, about 3 hours, about 2.5 hours, about 2 hours,about 1.5 hours, or about 1 hour (inclusive); between about 1 hour toabout 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16hours, about 14 hours, about 12 hours, about 10 hours, about 9 hours,about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4.5hours, about 4 hours, about 3.5 hours, about 3 hours, about 2.5 hours,about 2 hours, or about 1.5 hours (inclusive); between about 1.5 hoursto about 24 hours, about 22 hours, about 20 hours, about 18 hours, about16 hours, about 14 hours, about 12 hours, about 10 hours, about 9 hours,about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4.5hours, about 4 hours, about 3.5 hours, about 3 hours, about 2.5 hours,or about 2 hours (inclusive); between about 2 hours to about 24 hours,about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14hours, about 12 hours, about 10 hours, about 9 hours, about 8 hours,about 7 hours, about 6 hours, about 5 hours, about 4.5 hours, about 4hours, about 3.5 hours, about 3 hours, or about 2.5 hours (inclusive);between about 2.5 hours to about 24 hours, about 22 hours, about 20hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours,about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6hours, about 5 hours, about 4.5 hours, about 4 hours, about 3.5 hours,or about 3 hours (inclusive); between about 3 hours to about 24 hours,about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14hours, about 12 hours, about 10 hours, about 9 hours, about 8 hours,about 7 hours, about 6 hours, about 5 hours, about 4.5 hours, about 4hours, or about 3.5 hours (inclusive); between about 3.5 hours to about24 hours, about 22 hours, about 20 hours, about 18 hours, about 16hours, about 14 hours, about 12 hours, about 10 hours, about 9 hours,about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4.5hours, or about 4 hours (inclusive); between about 4 hours to about 24hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours,about 14 hours, about 12 hours, about 10 hours, about 9 hours, about 8hours, about 7 hours, about 6 hours, about 5 hours, or about 4.5 hours(inclusive); between about 4.5 hours to about 24 hours, about 22 hours,about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours,about 6 hours, or about 5 hours (inclusive); between about 5 hours toabout 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16hours, about 14 hours, about 12 hours, about 10 hours, about 9 hours,about 8 hours, about 7 hours, or about 6 hours (inclusive); betweenabout 6 hours to about 24 hours, about 22 hours, about 20 hours, about18 hours, about 16 hours, about 14 hours, about 12 hours, about 10hours, about 9 hours, about 8 hours, or about 7 hours (inclusive);between about 7 hours to about 24 hours, about 22 hours, about 20 hours,about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10hours, about 9 hours, or about 8 hours (inclusive); between about 8hours to about 24 hours, about 22 hours, about 20 hours, about 18 hours,about 16 hours, about 14 hours, about 12 hours, about 10 hours, or about9 hours (inclusive); between about 9 hours to about 24 hours, about 22hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours,about 12 hours, or about 10 hours (inclusive); between about 10 hours toabout 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16hours, about 14 hours, or about 12 hours (inclusive); between about 12hours to about 24 hours, about 22 hours, about 20 hours, about 18 hours,about 16 hours, or about 14 hours (inclusive); between about 14 hours toabout 24 hours, about 22 hours, about 20 hours, about 18 hours, or about16 hours (inclusive); between about 16 hours to about 24 hours, about 22hours, about 20 hours, or about 18 hours (inclusive); between about 18hours to about 24 hours, about 22 hours, or about 20 hours (inclusive);between about 20 hours to about 24 hours or about 22 hours (inclusive);or between about 22 hours to about 24 hours (inclusive).

Following the washing of the at least one chromatographic column orchromatographic membrane in the first MCCS that contains a resin that iscapable of performing the unit operation of capturing, the recombinanttherapeutic protein is eluted from the at least one chromatographiccolumn or chromatographic membrane by passing an elution buffer throughthe at least one chromatographic column or chromatographic membrane inthe first MCCS that contains a resin that is capable of performing theunit operation of capturing. The elution buffer can be passed throughthe at least one chromatography column or chromatographic membrane thatcontains a resin that is capable of performing the unit operation ofcapturing at a flow rate of between about 0.2 mL/minute to about 100L/minute (inclusive) (e.g., any of the subranges of this flow rate rangedescribed herein). The volume of elution buffer used to elute therecombinant therapeutic protein from each of the at least onechromatographic column or chromatographic membrane containing a resinthat is capable of performing the unit operation of purifying can be,e.g., between about 1× column volume (CV) to about 30×CV, about 28×CV,about 26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about16×CV, about 14×CV, about 13×CV, about 12×CV, about 11×CV, about 10×CV,about 9×CV, about 8×CV, about 7×CV, about 6×CV, about 5×CV, about 4×CV,or about 3×CV (inclusive); between about 2×CV to about 30×CV, about28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV,about 16×CV, about 14×CV, about 13×CV, about 12×CV, about 11×CV, about10×CV, about 9×CV, about 8×CV, about 7×CV, about 6×CV, about 5×CV, orabout 4×CV (inclusive); between about 3×CV to about 30×CV, about 28×CV,about 26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about16×CV, about 14×CV, about 13×CV, about 12×CV, about 11×CV, about 10×CV,about 9×CV, about 8×CV, about 7×CV, or about 5×CV (inclusive); betweenabout 4×CV to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about22×CV, about 20×CV, about 18×CV, about 16×CV, about 14×CV, about 13×CV,about 12×CV, about 11×CV, about 10×CV, about 9×CV, about 8×CV, about7×CV, or about 6×CV (inclusive); between about 5×CV to about 30×CV,about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV, about18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV, about 11×CV,about 10×CV, about 9×CV, about 8×CV, or about 7×CV (inclusive); betweenabout 6×CV to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about22×CV, about 20×CV, about 18×CV, about 16×CV, about 14×CV, about 13×CV,about 12×CV, about 11×CV, about 10×CV, about 9×CV, or about 8×CV(inclusive); between about 7×CV to about 30×CV, about 28×CV, about26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV,about 14×CV, about 13×CV, about 12×CV, about 11×CV, about 10×CV, orabout 9×CV (inclusive); between about 8×CV to about 30×CV, about 28×CV,about 26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about16×CV, about 14×CV, about 13×CV, about 12×CV, about 11×CV, or about10×CV (inclusive); between about 9 CV to about 30×CV, about 28×CV, about26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV,about 14×CV, about 13×CV, about 12×CV, or about 11×CV (inclusive);between about 10×CV to about 30×CV, about 28×CV, about 26×CV, about24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV, about 14×CV,about 13×CV, or about 12×CV (inclusive); between about 11×CV to about30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV,about 18×CV, about 16×CV, about 14×CV, or about 13×CV (inclusive);between about 12×CV to about 30×CV, about 28×CV, about 26×CV, about24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV, or about14×CV (inclusive); between about 14×CV to about 30×CV, about 28×CV,about 26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, orabout 16×CV (inclusive); between about 16×CV to about 30×CV, about28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV, or about18×CV (inclusive); between about 18×CV to about 30×CV, about 28×CV,about 26×CV, about 24×CV, about 22×CV, or about 20×CV (inclusive);between about 20×CV to about 30×CV, about 28×CV, about 26×CV, about24×CV, or about 22×CV (inclusive); between about 22×CV to about 30×CV,about 28×CV, about 26×CV, or about 24×CV (inclusive); between about24×CV to about 30×CV, about 28×CV, or about 26×CV (inclusive); betweenabout 26×CV to about 30×CV or about 28×CV (inclusive); or between about28×CV to about 30×CV (inclusive).

The total time of the eluting can be, e.g., between about 2 minutes toabout 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16hours, about 14 hours, about 12 hours, about 10 hours, about 7.5 hours,about 5 hours, about 2.5 hours, about 2.0 hours, about 1.5 hours, about1.5 hours, about 1.25 hours, about 1 hour, about 40 minutes, about 30minutes, about 20 minutes, about 10 minutes, or about 5 minutes,inclusive; between about 5 minutes to about 24 hours, about 22 hours,about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12hours, about 10 hours, about 7.5 hours, about 5 hours, about 2.5 hours,about 2.0 hours, about 1.5 hours, about 1.5 hours, about 1.25 hours,about 1 hour, about 40 minutes, about 30 minutes, about 20 minutes, orabout 10 minutes, inclusive; between about 10 minutes to about 24 hours,about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14hours, about 12 hours, about 10 hours, about 7.5 hours, about 5 hours,about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.5 hours,about 1.25 hours, about 1 hour, about 40 minutes, about 30 minutes, orabout 20 minutes, inclusive; between about 20 minutes to about 24 hours,about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14hours, about 12 hours, about 10 hours, about 7.5 hours, about 5 hours,about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.5 hours,about 1.25 hours, about 1 hour, about 40 minutes, or about 30 minutes,inclusive; between about 30 minutes to about 24 hours, about 22 hours,about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12hours, about 10 hours, about 7.5 hours, about 5 hours, about 2.5 hours,about 2.0 hours, about 1.5 hours, about 1.5 hours, about 1.25 hours,about 1 hour, or about 40 minutes, inclusive; between about 40 minutesto about 24 hours, about 22 hours, about 20 hours, about 18 hours, about16 hours, about 14 hours, about 12 hours, about 10 hours, about 7.5hours, about 5 hours, about 2.5 hours, about 2.0 hours, about 1.5 hours,about 1.5 hours, about 1.25 hours, or about 1 hour, inclusive; betweenabout 1 hour to about 24 hours, about 22 hours, about 20 hours, about 18hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours,about 7.5 hours, about 5 hours, about 2.5 hours, about 2.0 hours, about1.5 hours, about 1.5 hours, or about 1.25 hours, inclusive; betweenabout 1.25 hours to about 24 hours, about 22 hours, about 20 hours,about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10hours, about 7.5 hours, about 5 hours, about 2.5 hours, about 2.0 hours,about 1.5 hours, or about 1.5 hours, inclusive; between about 1.5 hourto about 24 hours, about 22 hours, about 20 hours, about 18 hours, about16 hours, about 14 hours, about 12 hours, about 10 hours, about 7.5hours, about 5 hours, about 2.5 hours, about 2.0 hours, or about 1.5hours, inclusive; between about 1.5 hours to about 24 hours, about 22hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours,about 12 hours, about 10 hours, about 7.5 hours, about 5 hours, about2.5 hours, or about 2.0 hours, inclusive; between about 2.0 hours toabout 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16hours, about 14 hours, about 12 hours, about 10 hours, about 7.5 hours,about 5 hours, or about 2.5 hours, inclusive; between about 2.5 hours toabout 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16hours, about 14 hours, about 12 hours, about 10 hours, about 7.5 hours,or about 5 hours, inclusive; between about 5 hours to about 24 hours,about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14hours, about 12 hours, about 10 hours, or about 7.5 hours, inclusive;between about 7.5 hours to about 24 hours, about 22 hours, about 20hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours,or about 10 hours, inclusive; between about 10 hours to about 24 hours,about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14hours, or about 12 hours, inclusive; between about 12 hours to about 24hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours,or about 14 hours, inclusive; between about 14 hours to about 24 hours,about 22 hours, about 20 hours, about 18 hours, or about 16 hours,inclusive; between about 16 hours to about 24 hours, about 22 hours,about 20 hours, or about 18 hours, inclusive; between about 18 hours toabout 24 hours, about 22 hours, or about 20 hours, inclusive; betweenabout 20 hours to about 24 hours or about 22 hours, inclusive; orbetween about 22 hours to about 24 hours, inclusive. Non-limitingexamples of elution buffers that can be used in these methods willdepend on the capture mechanism and/or the therapeutic protein. Forexample, an elution buffer can contain a different concentration of salt(e.g., increased salt concentration), a different pH (e.g., an increasedor decreased salt concentration), or a molecule that will compete withthe recombinant therapeutic protein for binding to the resin that iscapable of performing the unit operation of capturing. Examples of suchelution buffers for each exemplary capture mechanism described hereinare well known in the art.

Following the elution of the recombinant therapeutic protein from the atleast one chromatographic column or chromatographic membrane in thefirst MCCS that contains a resin that is capable of performing the unitoperation of capturing, and before the next volume of liquid culturemedium can be loaded onto the at least one chromatographic column orchromatographic membrane, the at least one chromatography column orchromatographic membrane must be equilibrated using an regenerationbuffer. The regeneration buffer can be passed through the at least onechromatography column or chromatographic membrane that contains a resinthat is capable of performing the unit operation of capturing at a flowrate of, e.g., between about 0.2 mL/minute to about 100 L/minute(inclusive) (e.g., any of the subranges of this flow rate rangedescribed herein). The volume of regeneration buffer used to equilibratethe at least one chromatography column or chromatographic membrane thatcontains a resin that is capable of performing the unit operation ofcapturing can be, e.g., between about 1× column volume (CV) to about30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV,about 18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV, about11×CV, about 10×CV, about 9×CV, about 8×CV, about 7×CV, about 6×CV,about 5×CV, about 4×CV, or about 3×CV (inclusive); between about 2×CV toabout 30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, about20×CV, about 18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV,about 11×CV, about 10×CV, about 9×CV, about 8×CV, about 7×CV, about6×CV, about 5×CV, or about 4×CV (inclusive); between about 3×CV to about30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV,about 18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV, about11×CV, about 10×CV, about 9×CV, about 8×CV, about 7×CV, or about 5×CV(inclusive); between about 4×CV to about 30×CV, about 28×CV, about26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV,about 14×CV, about 13×CV, about 12×CV, about 11×CV, about 10×CV, about9×CV, about 8×CV, about 7×CV, or about 6×CV (inclusive); between about5×CV to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV,about 20×CV, about 18×CV, about 16×CV, about 14×CV, about 13×CV, about12×CV, about 11×CV, about 10×CV, about 9×CV, about 8×CV, or about 7×CV(inclusive); between about 6×CV to about 30×CV, about 28×CV, about26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV,about 14×CV, about 13×CV, about 12×CV, about 11×CV, about 10×CV, about9×CV, or about 8×CV (inclusive); between about 7×CV to about 30×CV,about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV, about18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV, about 11×CV,about 10×CV, or about 9×CV (inclusive); between about 8×CV to about30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV,about 18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV, about11×CV, or about 10×CV (inclusive); between about 9 CV to about 30×CV,about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV, about18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV, or about11×CV (inclusive); between about 10×CV to about 30×CV, about 28×CV,about 26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about16×CV, about 14×CV, about 13×CV, or about 12×CV (inclusive); betweenabout 11×CV to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about22×CV, about 20×CV, about 18×CV, about 16×CV, about 14×CV, or about13×CV (inclusive); between about 12×CV to about 30×CV, about 28×CV,about 26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about16×CV, or about 14×CV (inclusive); between about 14×CV to about 30×CV,about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV, about18×CV, or about 16×CV (inclusive); between about 16×CV to about 30×CV,about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV, orabout 18×CV (inclusive); between about 18×CV to about 30×CV, about28×CV, about 26×CV, about 24×CV, about 22×CV, or about 20×CV(inclusive); between about 20×CV to about 30×CV, about 28×CV, about26×CV, about 24×CV, or about 22×CV (inclusive); between about 22×CV toabout 30×CV, about 28×CV, about 26×CV, or about 24×CV (inclusive);between about 24×CV to about 30×CV, about 28×CV, or about 26×CV(inclusive); between about 26×CV to about 30×CV or about 28×CV(inclusive); or between about 28×CV to about 30×CV (inclusive).

In some of the processes described herein, the first MCCS includes areservoir that holds a fluid containing the recombinant therapeuticprotein at low pH (e.g., a pH below 4.6, below 4.4, below 4.2, below4.0, below 3.8, below 3.6, below 3.4, below 3.2, or below 3.0) for,e.g., about 1 minute to 3.0 hours (e.g., about 1 hour), and inactivatesthe viruses present in a fluid containing the recombinant therapeuticprotein. An example of a reservoir that can be used to perform the unitoperation of inactivating viruses is a stir flask (e.g., 500-mL stirflask, e.g., a 500-mL stir flask with a programmed stir plate) that iscapable of holding a fluid containing a recombinant therapeutic proteinfor, e.g., about 1 minute to 1.5 hours, before the fluid containing therecombinant therapeutic protein is fed into the second MCCS. Thereservoir that is used to perform the unit operation of inactivation ofviruses can be a 500-mL stir flask with a programmed stir plate (e.g., astir plate programmed to mix (e.g., periodically mix) the fluid withinthe reservoir, e.g., every 4 hours). Another example of a reservoir thatcan be used to perform the unit operation of inactivation of viruses isa plastic bag (e.g., 500-mL plastic bag) that is capable of holding afluid containing a recombinant therapeutic protein for, e.g., about 1minute to 1.5 hours, before the fluid containing the recombinanttherapeutic protein is fed into the second MCCS. In some examples, thefluid containing the recombinant therapeutic protein can already have alow pH (e.g., a pH below 4.6, below 4.4, below 4.2, below 4.0, below3.8, below 3.6, below 3.4, below 3.2, or below 3.0) when it is fed intothe reservoir that is used to perform the unit operation of viralinactivation. Viral inactivation can also be performed using a tubularreactor (e.g., as described in Klutz et al., Chemical EngineeringProcessing: Process Intensification 102:88-101, 2016b; Klutz et al.,Chemical Engineering Research Design 95:22-33, 2015) or a coiled flowinverter (see, e.g., Klutz et al., Chemical Engineering Research Design95:22-33, 2015; and Klutz et al., Chemical Engineering Processing:Process Intensification 102:88-101, 2016b). As can be appreciated bythose skilled in the art, a variety of other means can be used toperform the unit operation of inactivating viruses. For example, UVirradiation (e.g., using UV-C light) of a fluid containing recombinanttherapeutic protein can also be used to perform the unit operation ofinactivating viruses (see, e.g., Lorenz et al., Biotechnol. Prog.25(2):476-482, 2009; Caillet-Fauquet et al., J. Virol. Methods118(2):131-139, 2004; Gunn et al., U.S. Pat. No. 6,586,172, 2003; Kaiseret al., U.S. Pat. No. 7,695,675, 2002; Bae et al., Kor. J. Microbiol.Biotechnol. 4:377-382, 2009; Li et al., Biologicals 33(2): 101-110,2005; and Wang et al., Vox Sang. 86(4):230-238, 2004). Non-limitingexamples of reservoirs that can be used to perform the unit operation ofinactivation of viruses present in a fluid containing the recombinanttherapeutic protein are described herein.

The first MCCS can include a PCCS containing four chromatographycolumns, where at least three of the four chromatography columns performthe unit operation of capturing the recombinant therapeutic protein fromthe liquid culture medium (e.g., using a first MCCS that includes any ofthe at least one chromatography columns that contain a resin that iscapable of performing the unit operation of capturing (e.g., any ofthose described herein)). In these examples, the fourth-column of thePCC can perform the unit operation of inactivating viruses in a fluidthat contains the recombinant therapeutic protein (e.g., any of theexemplary columns described herein that can be used to achieve viralinactivation of a fluid containing the recombinant therapeutic protein).

A fluid containing the recombinant therapeutic protein is continuouslyeluted from the first MCCS (e.g., the first PCC system), and iscontinuously fed into the second MCCS. The percent of the recombinanttherapeutic protein recovered in the eluate of the first MCCS (e.g., thefirst PCC system) can be, e.g., at least 70%, at least 72%, at least74%, at least 76%, at least 78%, at least 80%, at least 82%, at least84%, at least 86%, at least 88%, at least 90%, at least 92%, at least94%, at least 96%, or at least 98%). The eluate from the first MCCS(e.g., the first PCC system) can be fed into the second MCCS (e.g.,second PCC system) using a variety of means known in the art (e.g.,tubing). The eluate of the first MCCS (e.g., first PCC system) can befed into the second MCCS (e.g., second PCC system) at a flow rate of,e.g., between about 0.2 mL/minute to about 100 L/minute (inclusive)(e.g., any of the subranges of this flow rate range described herein).

Some processes described herein can further include a step of adjustingthe ionic concentration and/or pH of the eluate from the first MCCS(e.g., first PCC system) before it is fed into the second MCCS (e.g.,second PCC system). As described herein, the ionic concentration and/orpH of the eluate from the first MCCS (e.g., first PCC system) can beadjusted (before it is fed into the second MCCS) by adding a buffer tothe eluate (e.g., through the use of an in-line buffer adjustmentreservoir). The buffer can be added to the eluate from the first MCCS ata flow rate of, e.g., between about 0.2 mL/minute to about 100 L/minute(inclusive) (e.g., any of the subranges of this flow rate rangedescribed herein).

The processes described herein can further include a step of holding orstoring (and optionally also refrigerating) the eluate from the firstMCCS prior to feeding the eluate from the first MCCS into the secondMCCS. As described herein, this holding or storing step can be performedusing any of the reservoirs (e.g., back-up tanks) described herein.

The processes described herein can also include a step of filtering theeluate from the first MCCS before the eluate is fed into the secondMCCS. Any of the exemplary filters or methods for filtration describedherein can be used to filter the eluate from the first MCCS before theeluate is fed into the second MCCS.

Polishing and Purifying the Recombinant Therapeutic Protein

The processes described herein include a step of purifying and polishingthe recombinant therapeutic protein using a second MCCS, where theeluate from the MCC2 is a therapeutic protein drug substance. The secondMCCS can include at least one (e.g., two, three, or four) chromatographycolumn or chromatographic membrane that can be used to perform the unitoperation of purifying a recombinant therapeutic protein, and at leastone (e.g., two, three, or four) chromatography column or chromatographicmembrane that can be used to perform the unit operation of polishing therecombinant therapeutic protein.

The at least one chromatography column or chromatographic membrane thatcan be used to perform the unit operation of purifying the recombinanttherapeutic protein can contain a resin that utilizes a capturemechanism (e.g., any of the capture mechanisms described herein or knownin the art), or a resin that can be used to perform anion exchange,cation exchange, or molecular sieve chromatography. The at least onechromatography column or chromatographic membrane that can be used toperform the unit of operation of polishing the recombinant therapeuticprotein can contain a resin can be used to perform anion exchange,cation exchange, or molecular sieve chromatography (e.g., any of theexemplary resins for performing anion exchange, cation exchange, ormolecular sieve chromatography described herein or known in the art).

The size, shape, and volume of the at least one chromatography column orchromatography membrane that can be used to perform the unit ofoperation of purifying the recombinant therapeutic protein, and/or thesize and shape of the at least one chromatographic membrane that can beused to perform the unit of operation of polishing the recombinantmembrane can any of combination of the exemplary sizes, shapes, andvolumes of chromatography columns or chromatographic membranes describedherein.

As can be appreciated by one skilled in the art, the step of purifyingor polishing a recombinant therapeutic protein can, e.g., include thesteps of loading, washing, eluting, and equilibrating the at least onechromatography column or chromatographic membrane used to perform theunit of operation of purifying or polishing the recombinant therapeuticprotein. Typically, the elution buffer coming out of a chromatographycolumn or chromatographic membrane used to perform the unit operation ofpurifying contains the recombinant therapeutic protein. Typically, theloading and/or wash buffer coming out of a chromatography column orchromatographic membrane used to perform the unit operation of polishingcontains the recombinant therapeutic protein.

For example, the size of the at least one chromatography column orchromatographic membrane that can be used to perform unit operation ofpurifying the recombinant therapeutic protein can have a volume of,e.g., between about 2.0 mL to 200 L, about 180 L, about 160 L, about 140L, about 120 L, about 100 L, about 95 L, about 90 L, about 85 L, about80 L, about 75 L, about 70 L, about 65 L, about 60 L, about 55 L, about50 L, about 45 L, about 40 L, about 35 L, about 30 L, about 25 L, about20 L, about 15 L, about 10 L, about 5 L, about 1 L, about 950 mL, about900 mL, about 850 mL, about 800 mL, about 750 mL, about 700 mL, about650 mL, about 600 mL, about 550 mL, about 500 mL, about 450 mL, about400 mL, about 350 mL, about 300 mL, about 250 mL, about 200 mL, about180 mL, about 160 mL, about 140 mL, about 120 mL, about 100 mL, about 95mL, about 90 mL, about 85 mL, about 80 mL, about 75 mL, about 70 mL,about 65 mL, about 60 mL, about 55 mL, about 50 mL, about 45 mL, about40 mL, about 35 mL, about 30 mL, about 25 mL, about 20 mL, about 15 mL,about 10 mL, or about 5.0 mL (inclusive); between about 5.0 mL to 200 L,about 180 L, about 160 L, about 140 L, about 120 L, about 100 L, about95 L, about 90 L, about 85 L, about 80 L, about 75 L, about 70 L, about65 L, about 60 L, about 55 L, about 50 L, about 45 L, about 40 L, about35 L, about 30 L, about 25 L, about 20 L, about 15 L, about 10 L, about5 L, about 1 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL,about 750 mL, about 700 mL, about 650 mL, about 600 mL, about 550 mL,about 500 mL, about 450 mL, about 400 mL, about 350 mL, about 300 mL,about 250 mL, about 200 mL, about 180 mL, about 160 mL, about 140 mL,about 120 mL, about 100 mL, about 95 mL, about 90 mL, about 85 mL, about80 mL, about 75 mL, about 70 mL, about 65 mL, about 60 mL, about 55 mL,about 50 mL, about 45 mL, about 40 mL, about 35 mL, about 30 mL, about25 mL, about 20 mL, about 15 mL, or about 10 mL (inclusive); betweenabout 10 mL to 200 L, about 180 L, about 160 L, about 140 L, about 120L, about 100 L, about 95 L, about 90 L, about 85 L, about 80 L, about 75L, about 70 L, about 65 L, about 60 L, about 55 L, about 50 L, about 45L, about 40 L, about 35 L, about 30 L, about 25 L, about 20 L, about 15L, about 10 L, about 5 L, about 1 L, about 950 mL, about 900 mL, about850 mL, about 800 mL, about 750 mL, about 700 mL, about 650 mL, about600 mL, about 550 mL, about 500 mL, about 450 mL, about 400 mL, about350 mL, about 300 mL, about 250 mL, about 200 mL, about 180 mL, about160 mL, about 140 mL, about 120 mL, about 100 mL, about 95 mL, about 90mL, about 85 mL, about 80 mL, about 75 mL, about 70 mL, about 65 mL,about 60 mL, about 55 mL, about 50 mL, about 45 mL, about 40 mL, about35 mL, about 30 mL, about 25 mL, about 20 mL, or about 15 mL(inclusive); between about 15 mL to 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, about 95 L, about 90 L, about 85L, about 80 L, about 75 L, about 70 L, about 65 L, about 60 L, about 55L, about 50 L, about 45 L, about 40 L, about 35 L, about 30 L, about 25L, about 20 L, about 15 L, about 10 L, about 5 L, about 1 L, about 950mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL, about 700mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL, about 450mL, about 400 mL, about 350 mL, about 300 mL, about 250 mL, about 200mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL, about 100mL, about 95 mL, about 90 mL, about 85 mL, about 80 mL, about 75 mL,about 70 mL, about 65 mL, about 60 mL, about 55 mL, about 50 mL, about45 mL, about 40 mL, about 35 mL, about 30 mL, about 25 mL, or about 20mL (inclusive); between about 20 mL to 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, about 95 L, about 90 L, about 85L, about 80 L, about 75 L, about 70 L, about 65 L, about 60 L, about 55L, about 50 L, about 45 L, about 40 L, about 35 L, about 30 L, about 25L, about 20 L, about 15 L, about 10 L, about 5 L, about 1 L, about 950mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL, about 700mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL, about 450mL, about 400 mL, about 350 mL, about 300 mL, about 250 mL, about 200mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL, about 100mL, about 95 mL, about 90 mL, about 85 mL, about 80 mL, about 75 mL,about 70 mL, about 65 mL, about 60 mL, about 55 mL, about 50 mL, about45 mL, about 40 mL, about 35 mL, about 30 mL, or about 25 mL(inclusive); between about 25 mL to 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, about 95 L, about 90 L, about 85L, about 80 L, about 75 L, about 70 L, about 65 L, about 60 L, about 55L, about 50 L, about 45 L, about 40 L, about 35 L, about 30 L, about 25L, about 20 L, about 15 L, about 10 L, about 5 L, about 1 L, about 950mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL, about 700mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL, about 450mL, about 400 mL, about 350 mL, about 300 mL, about 250 mL, about 200mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL, about 100mL, about 95 mL, about 90 mL, about 85 mL, about 80 mL, about 75 mL,about 70 mL, about 65 mL, about 60 mL, about 55 mL, about 50 mL, about45 mL, about 40 mL, about 35 mL, or about 30 mL (inclusive); betweenabout 30 mL to 200 L, about 180 L, about 160 L, about 140 L, about 120L, about 100 L, about 95 L, about 90 L, about 85 L, about 80 L, about 75L, about 70 L, about 65 L, about 60 L, about 55 L, about 50 L, about 45L, about 40 L, about 35 L, about 30 L, about 25 L, about 20 L, about 15L, about 10 L, about 5 L, about 1 L, about 950 mL, about 900 mL, about850 mL, about 800 mL, about 750 mL, about 700 mL, about 650 mL, about600 mL, about 550 mL, about 500 mL, about 450 mL, about 400 mL, about350 mL, about 300 mL, about 250 mL, about 200 mL, about 180 mL, about160 mL, about 140 mL, about 120 mL, about 100 mL, about 95 mL, about 90mL, about 85 mL, about 80 mL, about 75 mL, about 70 mL, about 65 mL,about 60 mL, about 55 mL, about 50 mL, about 45 mL, about 40 mL, orabout 35 mL (inclusive); between about 35 mL to 200 L, about 180 L,about 160 L, about 140 L, about 120 L, about 100 L, about 95 L, about 90L, about 85 L, about 80 L, about 75 L, about 70 L, about 65 L, about 60L, about 55 L, about 50 L, about 45 L, about 40 L, about 35 L, about 30L, about 25 L, about 20 L, about 15 L, about 10 L, about 5 L, about 1 L,about 950 mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL,about 700 mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL,about 450 mL, about 400 mL, about 350 mL, about 300 mL, about 250 mL,about 200 mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL,about 100 mL, about 95 mL, about 90 mL, about 85 mL, about 80 mL, about75 mL, about 70 mL, about 65 mL, about 60 mL, about 55 mL, about 50 mL,about 45 mL, or about 40 mL (inclusive); between about 40 mL to 200 L,about 180 L, about 160 L, about 140 L, about 120 L, about 100 L, about95 L, about 90 L, about 85 L, about 80 L, about 75 L, about 70 L, about65 L, about 60 L, about 55 L, about 50 L, about 45 L, about 40 L, about35 L, about 30 L, about 25 L, about 20 L, about 15 L, about 10 L, about5 L, about 1 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL,about 750 mL, about 700 mL, about 650 mL, about 600 mL, about 550 mL,about 500 mL, about 450 mL, about 400 mL, about 350 mL, about 300 mL,about 250 mL, about 200 mL, about 180 mL, about 160 mL, about 140 mL,about 120 mL, about 100 mL, about 95 mL, about 90 mL, about 85 mL, about80 mL, about 75 mL, about 70 mL, about 65 mL, about 60 mL, about 55 mL,about 50 mL, or about 45 mL (inclusive); between about 45 mL to 200 L,about 180 L, about 160 L, about 140 L, about 120 L, about 100 L, about95 L, about 90 L, about 85 L, about 80 L, about 75 L, about 70 L, about65 L, about 60 L, about 55 L, about 50 L, about 45 L, about 40 L, about35 L, about 30 L, about 25 L, about 20 L, about 15 L, about 10 L, about5 L, about 1 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL,about 750 mL, about 700 mL, about 650 mL, about 600 mL, about 550 mL,about 500 mL, about 450 mL, about 400 mL, about 350 mL, about 300 mL,about 250 mL, about 200 mL, about 180 mL, about 160 mL, about 140 mL,about 120 mL, about 100 mL, about 95 mL, about 90 mL, about 85 mL, about80 mL, about 75 mL, about 70 mL, about 65 mL, about 60 mL, about 55 mL,or about 50 mL (inclusive); between about 50 mL to 200 L, about 180 L,about 160 L, about 140 L, about 120 L, about 100 L, about 95 L, about 90L, about 85 L, about 80 L, about 75 L, about 70 L, about 65 L, about 60L, about 55 L, about 50 L, about 45 L, about 40 L, about 35 L, about 30L, about 25 L, about 20 L, about 15 L, about 10 L, about 5 L, about 1 L,about 950 mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL,about 700 mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL,about 450 mL, about 400 mL, about 350 mL, about 300 mL, about 250 mL,about 200 mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL,about 100 mL, about 95 mL, about 90 mL, about 85 mL, about 80 mL, about75 mL, about 70 mL, about 65 mL, about 60 mL, or about 55 mL(inclusive); between about 55 mL to 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, about 95 L, about 90 L, about 85L, about 80 L, about 75 L, about 70 L, about 65 L, about 60 L, about 55L, about 50 L, about 45 L, about 40 L, about 35 L, about 30 L, about 25L, about 20 L, about 15 L, about 10 L, about 5 L, about 1 L, about 950mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL, about 700mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL, about 450mL, about 400 mL, about 350 mL, about 300 mL, about 250 mL, about 200mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL, about 100mL, about 95 mL, about 90 mL, about 85 mL, about 80 mL, about 75 mL,about 70 mL, about 65 mL, or about 60 mL (inclusive); between about 60mL to 200 L, about 180 L, about 160 L, about 140 L, about 120 L, about100 L, about 95 L, about 90 L, about 85 L, about 80 L, about 75 L, about70 L, about 65 L, about 60 L, about 55 L, about 50 L, about 45 L, about40 L, about 35 L, about 30 L, about 25 L, about 20 L, about 15 L, about10 L, about 5 L, about 1 L, about 950 mL, about 900 mL, about 850 mL,about 800 mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL,about 550 mL, about 500 mL, about 450 mL, about 400 mL, about 350 mL,about 300 mL, about 250 mL, about 200 mL, about 180 mL, about 160 mL,about 140 mL, about 120 mL, about 100 mL, about 95 mL, about 90 mL,about 85 mL, about 80 mL, about 75 mL, about 70 mL, or about 65 mL(inclusive); between about 65 mL to 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, about 95 L, about 90 L, about 85L, about 80 L, about 75 L, about 70 L, about 65 L, about 60 L, about 55L, about 50 L, about 45 L, about 40 L, about 35 L, about 30 L, about 25L, about 20 L, about 15 L, about 10 L, about 5 L, about 1 L, about 950mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL, about 700mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL, about 450mL, about 400 mL, about 350 mL, about 300 mL, about 250 mL, about 200mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL, about 100mL, about 95 mL, about 90 mL, about 85 mL, about 80 mL, about 75 mL, orabout 70 mL (inclusive); between about 70 mL to 200 L, about 180 L,about 160 L, about 140 L, about 120 L, about 100 L, about 95 L, about 90L, about 85 L, about 80 L, about 75 L, about 70 L, about 65 L, about 60L, about 55 L, about 50 L, about 45 L, about 40 L, about 35 L, about 30L, about 25 L, about 20 L, about 15 L, about 10 L, about 5 L, about 1 L,about 950 mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL,about 700 mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL,about 450 mL, about 400 mL, about 350 mL, about 300 mL, about 250 mL,about 200 mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL,about 100 mL, about 95 mL, about 90 mL, about 85 mL, about 80 mL, orabout 75 mL (inclusive); between about 75 mL to 200 L, about 180 L,about 160 L, about 140 L, about 120 L, about 100 L, about 95 L, about 90L, about 85 L, about 80 L, about 75 L, about 70 L, about 65 L, about 60L, about 55 L, about 50 L, about 45 L, about 40 L, about 35 L, about 30L, about 25 L, about 20 L, about 15 L, about 10 L, about 5 L, about 1 L,about 950 mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL,about 700 mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL,about 450 mL, about 400 mL, about 350 mL, about 300 mL, about 250 mL,about 200 mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL,about 100 mL, about 95 mL, about 90 mL, about 85 mL, or about 80 mL(inclusive); between about 80 mL to 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, about 95 L, about 90 L, about 85L, about 80 L, about 75 L, about 70 L, about 65 L, about 60 L, about 55L, about 50 L, about 45 L, about 40 L, about 35 L, about 30 L, about 25L, about 20 L, about 15 L, about 10 L, about 5 L, about 1 L, about 950mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL, about 700mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL, about 450mL, about 400 mL, about 350 mL, about 300 mL, about 250 mL, about 200mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL, about 100mL, about 95 mL, about 90 mL, or about 85 mL (inclusive); between about85 mL to 200 L, about 180 L, about 160 L, about 140 L, about 120 L,about 100 L, about 95 L, about 90 L, about 85 L, about 80 L, about 75 L,about 70 L, about 65 L, about 60 L, about 55 L, about 50 L, about 45 L,about 40 L, about 35 L, about 30 L, about 25 L, about 20 L, about 15 L,about 10 L, about 5 L, about 1 L, about 950 mL, about 900 mL, about 850mL, about 800 mL, about 750 mL, about 700 mL, about 650 mL, about 600mL, about 550 mL, about 500 mL, about 450 mL, about 400 mL, about 350mL, about 300 mL, about 250 mL, about 200 mL, about 180 mL, about 160mL, about 140 mL, about 120 mL, about 100 mL, about 95 mL, or about 90mL (inclusive); between about 90 mL to 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, about 95 L, about 90 L, about 85L, about 80 L, about 75 L, about 70 L, about 65 L, about 60 L, about 55L, about 50 L, about 45 L, about 40 L, about 35 L, about 30 L, about 25L, about 20 L, about 15 L, about 10 L, about 5 L, about 1 L, about 950mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL, about 700mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL, about 450mL, about 400 mL, about 350 mL, about 300 mL, about 250 mL, about 200mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL, about 100mL, or about 95 mL (inclusive); between about 95 mL to 200 L, about 180L, about 160 L, about 140 L, about 120 L, about 100 L, about 95 L, about90 L, about 85 L, about 80 L, about 75 L, about 70 L, about 65 L, about60 L, about 55 L, about 50 L, about 45 L, about 40 L, about 35 L, about30 L, about 25 L, about 20 L, about 15 L, about 10 L, about 5 L, about 1L, about 950 mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL,about 700 mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL,about 450 mL, about 400 mL, about 350 mL, about 300 mL, about 250 mL,about 200 mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL, orabout 100 mL (inclusive); between about 100 mL to 200 L, about 180 L,about 160 L, about 140 L, about 120 L, about 100 L, about 95 L, about 90L, about 85 L, about 80 L, about 75 L, about 70 L, about 65 L, about 60L, about 55 L, about 50 L, about 45 L, about 40 L, about 35 L, about 30L, about 25 L, about 20 L, about 15 L, about 10 L, about 5 L, about 1 L,about 950 mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL,about 700 mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL,about 450 mL, about 400 mL, about 350 mL, about 300 mL, about 250 mL,about 200 mL, about 180 mL, about 160 mL, about 140 mL, or about 120 mL(inclusive); between about 120 mL to 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, about 95 L, about 90 L, about 85L, about 80 L, about 75 L, about 70 L, about 65 L, about 60 L, about 55L, about 50 L, about 45 L, about 40 L, about 35 L, about 30 L, about 25L, about 20 L, about 15 L, about 10 L, about 5 L, about 1 L, about 950mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL, about 700mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL, about 450mL, about 400 mL, about 350 mL, about 300 mL, about 250 mL, about 200mL, about 180 mL, about 160 mL, or about 140 mL (inclusive); betweenabout 140 mL to 200 L, about 180 L, about 160 L, about 140 L, about 120L, about 100 L, about 95 L, about 90 L, about 85 L, about 80 L, about 75L, about 70 L, about 65 L, about 60 L, about 55 L, about 50 L, about 45L, about 40 L, about 35 L, about 30 L, about 25 L, about 20 L, about 15L, about 10 L, about 5 L, about 1 L, about 950 mL, about 900 mL, about850 mL, about 800 mL, about 750 mL, about 700 mL, about 650 mL, about600 mL, about 550 mL, about 500 mL, about 450 mL, about 400 mL, about350 mL, about 300 mL, about 250 mL, about 200 mL, about 180 mL, or about160 mL (inclusive); between about 160 mL to 200 L, about 180 L, about160 L, about 140 L, about 120 L, about 100 L, about 95 L, about 90 L,about 85 L, about 80 L, about 75 L, about 70 L, about 65 L, about 60 L,about 55 L, about 50 L, about 45 L, about 40 L, about 35 L, about 30 L,about 25 L, about 20 L, about 15 L, about 10 L, about 5 L, about 1 L,about 950 mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL,about 700 mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL,about 450 mL, about 400 mL, about 350 mL, about 300 mL, about 250 mL,about 200 mL, or about 180 mL (inclusive); between about 180 mL to 200L, about 180 L, about 160 L, about 140 L, about 120 L, about 100 L,about 95 L, about 90 L, about 85 L, about 80 L, about 75 L, about 70 L,about 65 L, about 60 L, about 55 L, about 50 L, about 45 L, about 40 L,about 35 L, about 30 L, about 25 L, about 20 L, about 15 L, about 10 L,about 5 L, about 1 L, about 950 mL, about 900 mL, about 850 mL, about800 mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL, about550 mL, about 500 mL, about 450 mL, about 400 mL, about 350 mL, about300 mL, about 250 mL, or about 200 mL (inclusive); between about 200 mLto 200 L, about 180 L, about 160 L, about 140 L, about 120 L, about 100L, about 95 L, about 90 L, about 85 L, about 80 L, about 75 L, about 70L, about 65 L, about 60 L, about 55 L, about 50 L, about 45 L, about 40L, about 35 L, about 30 L, about 25 L, about 20 L, about 15 L, about 10L, about 5 L, about 1 L, about 950 mL, about 900 mL, about 850 mL, about800 mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL, about550 mL, about 500 mL, about 450 mL, about 400 mL, about 350 mL, about300 mL, or about 250 mL (inclusive); between about 250 mL to 200 L,about 180 L, about 160 L, about 140 L, about 120 L, about 100 L, about95 L, about 90 L, about 85 L, about 80 L, about 75 L, about 70 L, about65 L, about 60 L, about 55 L, about 50 L, about 45 L, about 40 L, about35 L, about 30 L, about 25 L, about 20 L, about 15 L, about 10 L, about5 L, about 1 L, about 950 mL, about 900 mL, about 850 mL, about 800 mL,about 750 mL, about 700 mL, about 650 mL, about 600 mL, about 550 mL,about 500 mL, about 450 mL, about 400 mL, about 350 mL, or about 300 mL(inclusive); between about 300 mL to 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, about 95 L, about 90 L, about 85L, about 80 L, about 75 L, about 70 L, about 65 L, about 60 L, about 55L, about 50 L, about 45 L, about 40 L, about 35 L, about 30 L, about 25L, about 20 L, about 15 L, about 10 L, about 5 L, about 1 L, about 950mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL, about 700mL, about 650 mL, about 600 mL, about 550 mL, about 500 mL, about 450mL, about 400 mL, or about 350 mL (inclusive); between about 350 mL to200 L, about 180 L, about 160 L, about 140 L, about 120 L, about 100 L,about 95 L, about 90 L, about 85 L, about 80 L, about 75 L, about 70 L,about 65 L, about 60 L, about 55 L, about 50 L, about 45 L, about 40 L,about 35 L, about 30 L, about 25 L, about 20 L, about 15 L, about 10 L,about 5 L, about 1 L, about 950 mL, about 900 mL, about 850 mL, about800 mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL, about550 mL, about 500 mL, about 450 mL, or about 400 mL (inclusive); betweenabout 400 mL to 200 L, about 180 L, about 160 L, about 140 L, about 120L, about 100 L, about 95 L, about 90 L, about 85 L, about 80 L, about 75L, about 70 L, about 65 L, about 60 L, about 55 L, about 50 L, about 45L, about 40 L, about 35 L, about 30 L, about 25 L, about 20 L, about 15L, about 10 L, about 5 L, about 1 L, about 950 mL, about 900 mL, about850 mL, about 800 mL, about 750 mL, about 700 mL, about 650 mL, about600 mL, about 550 mL, about 500 mL, or about 450 mL (inclusive); betweenabout 450 mL to 200 L, about 180 L, about 160 L, about 140 L, about 120L, about 100 L, about 95 L, about 90 L, about 85 L, about 80 L, about 75L, about 70 L, about 65 L, about 60 L, about 55 L, about 50 L, about 45L, about 40 L, about 35 L, about 30 L, about 25 L, about 20 L, about 15L, about 10 L, about 5 L, about 1 L, about 950 mL, about 900 mL, about850 mL, about 800 mL, about 750 mL, about 700 mL, about 650 mL, about600 mL, about 550 mL, or about 500 mL (inclusive); between about 500 mLto 200 L, about 180 L, about 160 L, about 140 L, about 120 L, about 100L, about 95 L, about 90 L, about 85 L, about 80 L, about 75 L, about 70L, about 65 L, about 60 L, about 55 L, about 50 L, about 45 L, about 40L, about 35 L, about 30 L, about 25 L, about 20 L, about 15 L, about 10L, about 5 L, about 1 L, about 950 mL, about 900 mL, about 850 mL, about800 mL, about 750 mL, about 700 mL, about 650 mL, about 600 mL, or about550 mL (inclusive); between about 550 mL to 200 L, about 180 L, about160 L, about 140 L, about 120 L, about 100 L, about 95 L, about 90 L,about 85 L, about 80 L, about 75 L, about 70 L, about 65 L, about 60 L,about 55 L, about 50 L, about 45 L, about 40 L, about 35 L, about 30 L,about 25 L, about 20 L, about 15 L, about 10 L, about 5 L, about 1 L,about 950 mL, about 900 mL, about 850 mL, about 800 mL, about 750 mL,about 700 mL, about 650 mL, or about 600 mL (inclusive); between about600 mL to 200 L, about 180 L, about 160 L, about 140 L, about 120 L,about 100 L, about 95 L, about 90 L, about 85 L, about 80 L, about 75 L,about 70 L, about 65 L, about 60 L, about 55 L, about 50 L, about 45 L,about 40 L, about 35 L, about 30 L, about 25 L, about 20 L, about 15 L,about 10 L, about 5 L, about 1 L, about 950 mL, about 900 mL, about 850mL, about 800 mL, about 750 mL, about 700 mL, or about 650 mL; betweenabout 650 mL to 200 L, about 180 L, about 160 L, about 140 L, about 120L, about 100 L, about 95 L, about 90 L, about 85 L, about 80 L, about 75L, about 70 L, about 65 L, about 60 L, about 55 L, about 50 L, about 45L, about 40 L, about 35 L, about 30 L, about 25 L, about 20 L, about 15L, about 10 L, about 5 L, about 1 L, about 950 mL, about 900 mL, about850 mL, about 800 mL, about 750 mL, or about 700 mL (inclusive); betweenabout 700 mL to 200 L, about 180 L, about 160 L, about 140 L, about 120L, about 100 L, about 95 L, about 90 L, about 85 L, about 80 L, about 75L, about 70 L, about 65 L, about 60 L, about 55 L, about 50 L, about 45L, about 40 L, about 35 L, about 30 L, about 25 L, about 20 L, about 15L, about 10 L, about 5 L, about 1 L, about 950 mL, about 900 mL, about850 mL, about 800 mL, or about 750 mL (inclusive); between about 750 mLto 200 L, about 180 L, about 160 L, about 140 L, about 120 L, about 100L, about 95 L, about 90 L, about 85 L, about 80 L, about 75 L, about 70L, about 65 L, about 60 L, about 55 L, about 50 L, about 45 L, about 40L, about 35 L, about 30 L, about 25 L, about 20 L, about 15 L, about 10L, about 5 L, about 1 L, about 950 mL, about 900 mL, about 850 mL, orabout 800 mL (inclusive); between about 800 mL to 200 L, about 180 L,about 160 L, about 140 L, about 120 L, about 100 L, about 95 L, about 90L, about 85 L, about 80 L, about 75 L, about 70 L, about 65 L, about 60L, about 55 L, about 50 L, about 45 L, about 40 L, about 35 L, about 30L, about 25 L, about 20 L, about 15 L, about 10 L, about 5 L, about 1 L,about 950 mL, about 900 mL, or about 850 mL (inclusive); between about850 mL to 200 L, about 180 L, about 160 L, about 140 L, about 120 L,about 100 L, about 95 L, about 90 L, about 85 L, about 80 L, about 75 L,about 70 L, about 65 L, about 60 L, about 55 L, about 50 L, about 45 L,about 40 L, about 35 L, about 30 L, about 25 L, about 20 L, about 15 L,about 10 L, about 5 L, about 1 L, about 950 mL, or about 900 mL(inclusive); between about 900 mL to 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, about 95 L, about 90 L, about 85L, about 80 L, about 75 L, about 70 L, about 65 L, about 60 L, about 55L, about 50 L, about 45 L, about 40 L, about 35 L, about 30 L, about 25L, about 20 L, about 15 L, about 10 L, about 5 L, about 1 L, or about950 mL (inclusive); between about 950 mL to 200 L, about 180 L, about160 L, about 140 L, about 120 L, about 100 L, about 95 L, about 90 L,about 85 L, about 80 L, about 75 L, about 70 L, about 65 L, about 60 L,about 55 L, about 50 L, about 45 L, about 40 L, about 35 L, about 30 L,about 25 L, about 20 L, about 15 L, about 10 L, about 5 L, or about 1 L(inclusive); between about 1 L to 200 L, about 180 L, about 160 L, about140 L, about 120 L, about 100 L, about 95 L, about 90 L, about 85 L,about 80 L, about 75 L, about 70 L, about 65 L, about 60 L, about 55 L,about 50 L, about 45 L, about 40 L, about 35 L, about 30 L, about 25 L,about 20 L, about 15 L, about 10 L, or about 5 L (inclusive); betweenabout 5 L to 200 L, about 180 L, about 160 L, about 140 L, about 120 L,about 100 L, about 95 L, about 90 L, about 85 L, about 80 L, about 75 L,about 70 L, about 65 L, about 60 L, about 55 L, about 50 L, about 45 L,about 40 L, about 35 L, about 30 L, about 25 L, about 20 L, about 15 L,or about 10 L (inclusive); between about 10 L to 200 L, about 180 L,about 160 L, about 140 L, about 120 L, about 100 L, about 95 L, about 90L, about 85 L, about 80 L, about 75 L, about 70 L, about 65 L, about 60L, about 55 L, about 50 L, about 45 L, about 40 L, about 35 L, about 30L, about 25 L, about 20 L, or about 15 L (inclusive); between about 15 Lto 200 L, about 180 L, about 160 L, about 140 L, about 120 L, about 100L, about 95 L, about 90 L, about 85 L, about 80 L, about 75 L, about 70L, about 65 L, about 60 L, about 55 L, about 50 L, about 45 L, about 40L, about 35 L, about 30 L, about 25 L, or about 20 L (inclusive);between about 20 L to 200 L, about 180 L, about 160 L, about 140 L,about 120 L, about 100 L, about 95 L, about 90 L, about 85 L, about 80L, about 75 L, about 70 L, about 65 L, about 60 L, about 55 L, about 50L, about 45 L, about 40 L, about 35 L, about 30 L, or about 25 L(inclusive); between about 25 L to 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, about 95 L, about 90 L, about 85L, about 80 L, about 75 L, about 70 L, about 65 L, about 60 L, about 55L, about 50 L, about 45 L, about 40 L, about 35 L, or about 30 L(inclusive); between about 30 L to 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, about 95 L, about 90 L, about 85L, about 80 L, about 75 L, about 70 L, about 65 L, about 60 L, about 55L, about 50 L, about 45 L, about 40 L, or about 35 L (inclusive);between about 35 L to 200 L, about 180 L, about 160 L, about 140 L,about 120 L, about 100 L, about 95 L, about 90 L, about 85 L, about 80L, about 75 L, about 70 L, about 65 L, about 60 L, about 55 L, about 50L, about 45 L, or about 40 L (inclusive); between about 40 L to 200 L,about 180 L, about 160 L, about 140 L, about 120 L, about 100 L, about95 L, about 90 L, about 85 L, about 80 L, about 75 L, about 70 L, about65 L, about 60 L, about 55 L, about 50 L, or about 45 L (inclusive);between about 45 L to 200 L, about 180 L, about 160 L, about 140 L,about 120 L, about 100 L, about 95 L, about 90 L, about 85 L, about 80L, about 75 L, about 70 L, about 65 L, about 60 L, about 55 L, or about50 L (inclusive); between about 50 L to 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, about 95 L, about 90 L, about 85L, about 80 L, about 75 L, about 70 L, about 65 L, about 60 L, or about55 L (inclusive); between about 55 L to 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, about 95 L, about 90 L, about 85L, about 80 L, about 75 L, about 70 L, about 65 L, or about 60 L(inclusive); between about 60 L to 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, about 95 L, about 90 L, about 85L, about 80 L, about 75 L, about 70 L, or about 65 L (inclusive);between about 65 L to 200 L, about 180 L, about 160 L, about 140 L,about 120 L, about 100 L, about 95 L, about 90 L, about 85 L, about 80L, about 75 L, or about 70 L (inclusive); between about 70 L to 200 L,about 180 L, about 160 L, about 140 L, about 120 L, about 100 L, about95 L, about 90 L, about 85 L, about 80 L, or about 75 L (inclusive);between about 75 L to 200 L, about 180 L, about 160 L, about 140 L,about 120 L, about 100 L, about 95 L, about 90 L, about 85 L, or about80 L (inclusive); between about 80 L to 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, about 95 L, about 90 L, or about85 L (inclusive); between about 85 L to 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, about 95 L, or about 90 L(inclusive); between about 90 L to 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, or about 95 L (inclusive);between about 95 L to 200 L, about 180 L, about 160 L, about 140 L,about 120 L, or about 100 L (inclusive); between about 100 L to 200 L,about 180 L, about 160 L, about 140 L, or about 120 L (inclusive);between about 120 L to 200 L, about 180 L, about 160 L, or about 140 L(inclusive); between about 140 L to 200 L, about 180 L, or about 160 L(inclusive); between about 160 L to about 200 L or about 180 L(inclusive); or between about 180 L to about 200 L (inclusive).

The flow rate of the fluid containing the recombinant therapeuticprotein as it is loaded onto the at least one chromatography column orat least one chromatographic that can be used to perform the unitoperation of purifying the recombinant therapeutic protein can be, e.g.,between about 0.1 mL/minute to about 100 L/minute, about 80 L/minute,about 60 L/minute, about 40 L/minute, about 20 L/minute, about 10L/minute, about 5 L/minute, about 1 L/minute, about 800 mL/minute, about600 mL/minute, about 400 mL/minute, about 200 mL/minute, about 100mL/minute, about 25 mL/minute, about 12.5 mL/minute, about 10.0mL/minute, about 8.0 mL/minute, about 6 mL/minute, about 4 mL/minute,about 3 mL/minute, about 2 mL/minute, about 1 mL/minute, or about 0.5mL/minute (inclusive); between about 0.5 mL/minute to about 100L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute, about20 L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute,about 800 mL/minute, about 600 mL/minute, about 400 mL/minute, about 200mL/minute, about 100 mL/minute, about 25 mL/minute, about 12.5mL/minute, about 10.0 mL/minute, about 8.0 mL/minute, about 6 mL/minute,about 4 mL/minute, about 3 mL/minute, about 2 mL/minute, or about 1mL/minute (inclusive); between about 1 mL/minute to about 100 L/minute,about 80 L/minute, about 60 L/minute, about 40 L/minute, about 20L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about800 mL/minute, about 600 mL/minute, about 400 mL/minute, about 200mL/minute, about 100 mL/minute, about 25 mL/minute, about 12.5mL/minute, about 10.0 mL/minute, about 8.0 mL/minute, about 6 mL/minute,about 4 mL/minute, about 3 mL/minute, or about 2 mL/minute (inclusive);between about 2 mL/minute to about 100 L/minute, about 80 L/minute,about 60 L/minute, about 40 L/minute, about 20 L/minute, about 10L/minute, about 5 L/minute, about 1 L/minute, about 800 mL/minute, about600 mL/minute, about 400 mL/minute, about 200 mL/minute, about 100mL/minute, about 25 mL/minute, about 12.5 mL/minute, about 10.0mL/minute, about 8.0 mL/minute, about 6 mL/minute, about 4 mL/minute, orabout 3 mL/minute (inclusive); between about 3 mL/minute to about 100L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute, about20 L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute,about 800 mL/minute, about 600 mL/minute, about 400 mL/minute, about 200mL/minute, about 100 mL/minute, about 25 mL/minute, about 12.5mL/minute, about 10.0 mL/minute, about 8.0 mL/minute, about 6 mL/minute,or about 4 mL/minute (inclusive); between about 4 mL/minute to about 100L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute, about20 L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute,about 800 mL/minute, about 600 mL/minute, about 400 mL/minute, about 200mL/minute, about 100 mL/minute, about 25 mL/minute, about 12.5mL/minute, about 10.0 mL/minute, about 8.0 mL/minute, or about 6mL/minute (inclusive); between about 6 mL/minute to about 100 L/minute,about 80 L/minute, about 60 L/minute, about 40 L/minute, about 20L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about800 mL/minute, about 600 mL/minute, about 400 mL/minute, about 200mL/minute, about 100 mL/minute, about 25 mL/minute, about 12.5mL/minute, about 10.0 mL/minute, or about 8.0 mL/minute (inclusive);between about 8 mL/minute to about 100 L/minute, about 80 L/minute,about 60 L/minute, about 40 L/minute, about 20 L/minute, about 10L/minute, about 5 L/minute, about 1 L/minute, about 800 mL/minute, about600 mL/minute, about 400 mL/minute, about 200 mL/minute, about 100mL/minute, about 25 mL/minute, about 12.5 mL/minute, or about 10.0mL/minute (inclusive); between about 10 mL/minute to about 100 L/minute,about 80 L/minute, about 60 L/minute, about 40 L/minute, about 20L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about800 mL/minute, about 600 mL/minute, about 400 mL/minute, about 200mL/minute, about 100 mL/minute, about 25 mL/minute, or about 12.5mL/minute (inclusive); between about 12.5 mL/minute to about 100L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute, about20 L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute,about 800 mL/minute, about 600 mL/minute, about 400 mL/minute, about 200mL/minute, about 100 mL/minute, or about 25 mL/minute (inclusive);between about 25 mL/minute to about 100 L/minute, about 80 L/minute,about 60 L/minute, about 40 L/minute, about 20 L/minute, about 10L/minute, about 5 L/minute, about 1 L/minute, about 800 mL/minute, about600 mL/minute, about 400 mL/minute, about 200 mL/minute, or about 100mL/minute (inclusive); between about 100 mL/minute to about 100L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute, about20 L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute,about 800 mL/minute, about 600 mL/minute, about 400 mL/minute, or about200 mL/minute (inclusive); between about 200 mL/minute to about 100L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute, about20 L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute,about 800 mL/minute, about 600 mL/minute, or about 400 mL/minute(inclusive); between about 400 mL/minute to about 100 L/minute, about 80L/minute, about 60 L/minute, about 40 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 800 mL/minute, orabout 600 mL/minute (inclusive); between about 600 mL/minute to about100 L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, or about 800 mL/minute (inclusive); between about 800mL/minute to about 100 L/minute, about 80 L/minute, about 60 L/minute,about 40 L/minute, about 20 L/minute, about 10 L/minute, about 5L/minute, or about 1 L/minute (inclusive); between about 1 L/minute toabout 100 L/minute, about 80 L/minute, about 60 L/minute, about 40L/minute, about 20 L/minute, about 10 L/minute, or about 5 L/minute(inclusive); between about 5 L/minute to about 100 L/minute, about 80L/minute, about 60 L/minute, about 40 L/minute, about 20 L/minute, orabout 10 L/minute (inclusive); between about 10 L/minute to about 100L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute, orabout 20 L/minute (inclusive); between about 20 L/minute to about 100L/minute, about 80 L/minute, about 60 L/minute, or about 40 L/minute(inclusive); between about 40 L/minute to about 100 L/minute, about 80L/minute, or about 60 L/minute (inclusive); between about 60 L/minute to100 L/minute or about 80 L/minute (inclusive); or between about 80L/minute to about 100 L/minute (inclusive)

The concentration of the recombinant therapeutic protein in the fluidloaded onto the at least one chromatography column or chromatographicmembrane that can be used to perform the unit operation of purifying therecombinant therapeutic protein can be, e.g., between about 0.05 mg/mLto about 100 mg/mL recombinant protein (e.g., between about 0.1 mg/mL toabout 90 mg/mL, between about 0.1 mg/mL to about 80 mg/mL, between about0.1 mg/mL to about 70 mg/mL, between about 0.1 mg/mL to about 60 mg/mL,between about 0.1 mg/mL to about 50 mg/mL, between about 0.1 mg/mL toabout 40 mg/mL, between about 0.1 mg/mL to about 30 mg/mL, between about0.1 mg/mL to about 20 mg/mL, between 0.5 mg/mL to about 20 mg/mL,between about 0.1 mg/mL to about 15 mg/mL, between about 0.5 mg/mL toabout 15 mg/mL, between about 0.1 mg/mL to about 10 mg/mL, or betweenabout 0.5 mg/mL to about 10 mg/mL recombinant therapeutic protein). Theresin in the at least one chromatography column or chromatographicmembrane used to perform unit operation of purifying can be a resin thatcan be used to perform anion exchange or cation exchange chromatography.The resin in the at least one chromatography column or chromatographicmembrane that is used to perform the unit operation of purifying can bea cationic exchange resin (e.g., Capto-S resin, GE Healthcare LifeSciences, Piscataway, N.J.).

Following the loading of the recombinant therapeutic protein onto the atleast one chromatographic column or chromatographic membrane in thesecond MCCS that can be used to perform the unit operation of purifyingthe recombinant therapeutic protein, the at least one chromatographiccolumn or chromatographic membrane is washed with at least one washingbuffer. As can be appreciated in the art, the at least one (e.g., two,three, or four) washing buffer is meant to elute all proteins that arenot the recombinant therapeutic protein from the at least onechromatography column or chromatographic membrane, while not disturbingthe interaction of the recombinant therapeutic protein with the resin orotherwise eluting the recombinant therapeutic protein.

The wash buffer can be passed through the at least one chromatographycolumn or chromatographic membrane at a flow rate of, e.g., betweenabout 0.2 mL/minute to about 100 L/minute (inclusive) (e.g., any of thesubranges of this flow rate range described herein). The volume of washbuffer used (e.g., combined total volume of wash buffer used when morethan one wash buffer is used) can be, e.g., between about 1× columnvolume (CV) to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about22×CV, about 20×CV, about 18×CV, about 16×CV, about 14×CV, about 13×CV,about 12×CV, about 11×CV, about 10×CV, about 9×CV, about 8×CV, about7×CV, about 6×CV, about 5×CV, about 4×CV, or about 3×CV (inclusive);between about 2×CV to about 30×CV, about 28×CV, about 26×CV, about24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV, about 14×CV,about 13×CV, about 12×CV, about 11×CV, about 10×CV, about 9×CV, about8×CV, about 7×CV, about 6×CV, about 5×CV, or about 4×CV (inclusive);between about 3×CV to about 30×CV, about 28×CV, about 26×CV, about24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV, about 14×CV,about 13×CV, about 12×CV, about 11×CV, about 10×CV, about 9×CV, about8×CV, about 7×CV, or about 5×CV (inclusive); between about 4×CV to about30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV,about 18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV, about11×CV, about 10×CV, about 9×CV, about 8×CV, about 7×CV, or about 6×CV(inclusive); between about 5×CV to about 30×CV, about 28×CV, about26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV,about 14×CV, about 13×CV, about 12×CV, about 11×CV, about 10×CV, about9×CV, about 8×CV, or about 7×CV (inclusive); between about 6×CV to about30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV,about 18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV, about11×CV, about 10×CV, about 9×CV, or about 8×CV (inclusive); between about7×CV to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV,about 20×CV, about 18×CV, about 16×CV, about 14×CV, about 13×CV, about12×CV, about 11×CV, about 10×CV, or about 9×CV (inclusive); betweenabout 8×CV to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about22×CV, about 20×CV, about 18×CV, about 16×CV, about 14×CV, about 13×CV,about 12×CV, about 11×CV, or about 10×CV (inclusive); between about 9 CVto about 30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV,about 20×CV, about 18×CV, about 16×CV, about 14×CV, about 13×CV, about12×CV, or about 11×CV (inclusive); between about 10×CV to about 30×CV,about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV, about18×CV, about 16×CV, about 14×CV, about 13×CV, or about 12×CV(inclusive); between about 11×CV to about 30×CV, about 28×CV, about26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV,about 14×CV, or about 13×CV (inclusive); between about 12×CV to about30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV,about 18×CV, about 16×CV, or about 14×CV (inclusive); between about14×CV to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about22×CV, about 20×CV, about 18×CV, or about 16×CV (inclusive); betweenabout 16×CV to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about22×CV, about 20×CV, or about 18×CV (inclusive); between about 18×CV toabout 30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, orabout 20×CV (inclusive); between about 20×CV to about 30×CV, about28×CV, about 26×CV, about 24×CV, or about 22×CV (inclusive); betweenabout 22×CV to about 30×CV, about 28×CV, about 26×CV, or about 24×CV(inclusive); between about 24×CV to about 30×CV, about 28×CV, or about26×CV (inclusive); between about 26×CV to about 30×CV or about 28×CV(inclusive); or between about 28×CV to about 30×CV (inclusive). Thetotal time of the washing can be, e.g., between about 2 minutes to about48 hours, about 40 hours, about 36 hours, about 32 hours, about 28hours, about 24 hours, about 20 hours, about 16 hours, about 12 hours,about 8 hours, about 4 hours, about 2.5 hours, about 2.0 hours, about1.5 hours, about 1.25 hours, about 1 hour, about 30 minutes, about 20minutes, about 10 minutes, or about 5 minutes (inclusive); between about5 minutes to about 48 hours, about 40 hours, about 36 hours, about 32hours, about 28 hours, about 24 hours, about 20 hours, about 16 hours,about 12 hours, about 8 hours, about 4 hours, about 2.5 hours, about 2.0hours, about 1.5 hours, about 1.25 hours, about 1 hour, about 30minutes, about 20 minutes, or about 10 minutes (inclusive); betweenabout 10 minutes to about 48 hours, about 40 hours, about 36 hours,about 32 hours, about 28 hours, about 24 hours, about 20 hours, about 16hours, about 12 hours, about 8 hours, about 4 hours, about 2.5 hours,about 2.0 hours, about 1.5 hours, about 1.25 hours, about 1 hour, about30 minutes, or about 20 minutes (inclusive); between about 20 minutes toabout 48 hours, about 40 hours, about 36 hours, about 32 hours, about 28hours, about 24 hours, about 20 hours, about 16 hours, about 12 hours,about 8 hours, about 4 hours, about 2.5 hours, about 2.0 hours, about1.5 hours, about 1.25 hours, about 1 hour, or about 30 minutes(inclusive); between about 30 minutes to about 48 hours, about 40 hours,about 36 hours, about 32 hours, about 28 hours, about 24 hours, about 20hours, about 16 hours, about 12 hours, about 8 hours, about 4 hours,about 2.5 hours, about 2.0 hours, about 1.5 hours, about 1.25 hours, orabout 1 hour (inclusive); between about 1 hour to about 48 hours, about40 hours, about 36 hours, about 32 hours, about 28 hours, about 24hours, about 20 hours, about 16 hours, about 12 hours, about 8 hours,about 4 hours, about 2.5 hours, about 2.0 hours, about 1.5 hours, orabout 1.25 hours (inclusive); between about 1.25 hours to about 48hours, about 40 hours, about 36 hours, about 32 hours, about 28 hours,about 24 hours, about 20 hours, about 16 hours, about 12 hours, about 8hours, about 4 hours, about 2.5 hours, about 2.0 hours, or about 1.5hours (inclusive); between about 1.5 hours to about 48 hours, about 40hours, about 36 hours, about 32 hours, about 28 hours, about 24 hours,about 20 hours, about 16 hours, about 12 hours, about 8 hours, about 4hours, about 2.5 hours, or about 2.0 hours (inclusive); between about2.0 hours to about 48 hours, about 40 hours, about 36 hours, about 32hours, about 28 hours, about 24 hours, about 20 hours, about 16 hours,about 12 hours, about 8 hours, about 4 hours, or about 2.5 hours(inclusive); between about 2.5 hours to about 48 hours, about 40 hours,about 36 hours, about 32 hours, about 28 hours, about 24 hours, about 20hours, about 16 hours, about 12 hours, about 8 hours, or about 4 hours(inclusive); between about 4 hours to about 48 hours, about 40 hours,about 36 hours, about 32 hours, about 28 hours, about 24 hours, about 20hours, about 16 hours, about 12 hours, or about 8 hours (inclusive);between about 8 hours to about 48 hours, about 40 hours, about 36 hours,about 32 hours, about 28 hours, about 24 hours, about 20 hours, about 16hours, or about 12 hours (inclusive); between about 12 hours to about 48hours, about 40 hours, about 36 hours, about 32 hours, about 28 hours,about 24 hours, about 20 hours, or about 16 hours (inclusive); betweenabout 16 hours to about 48 hours, about 40 hours, about 36 hours, about32 hours, about 28 hours, about 24 hours, or about 20 hours (inclusive);between about 20 hours to about 48 hours, about 40 hours, about 36hours, about 32 hours, about 28 hours, or about 24 hours (inclusive);between about 24 hours to about 48 hours, about 40 hours, about 36hours, about 32 hours, or about 28 hours (inclusive); between about 28hours to about 48 hours, about 40 hours, about 36 hours, or about 32hours (inclusive); between about 32 hours to about 48 hours, about 40hours, or about 36 hours (inclusive); between about 36 hours to about 48hours or about 40 hours (inclusive); or between about 40 hours to about48 hours (inclusive).

Following the washing of the at least one chromatographic column orchromatographic membrane in the second MCCS that can be used to performthe unit operation of purifying the recombinant therapeutic protein, therecombinant therapeutic protein is eluted from the at least onechromatographic column or chromatographic membrane by passing an elutionbuffer through the at least one chromatographic column orchromatographic membrane in the second MCCS that can be used to performthe unit operation of purifying the recombinant therapeutic protein. Theelution buffer can be passed through the at least one chromatographycolumn or chromatographic membrane that can be used to perform the unitoperation of purifying the recombinant therapeutic protein at a flowrate of, e.g., between about 0.2 mL/minute to about 100 L/minute(inclusive) (e.g., any of the subranges of this flow rate rangedescribed herein). The volume of elution buffer used to elute therecombinant therapeutic protein from each the at least onechromatographic column or chromatographic membrane that can be used toperform the unit operation of purifying the recombinant therapeuticprotein can be, e.g., between about 1× column volume (CV) to about30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV,about 18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV, about11×CV, about 10×CV, about 9×CV, about 8×CV, about 7×CV, about 6×CV,about 5×CV, about 4×CV, or about 3×CV (inclusive); between about 2×CV toabout 30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, about20×CV, about 18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV,about 11×CV, about 10×CV, about 9×CV, about 8×CV, about 7×CV, about6×CV, about 5×CV, or about 4×CV (inclusive); between about 3×CV to about30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV,about 18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV, about11×CV, about 10×CV, about 9×CV, about 8×CV, about 7×CV, or about 5×CV(inclusive); between about 4×CV to about 30×CV, about 28×CV, about26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV,about 14×CV, about 13×CV, about 12×CV, about 11×CV, about 10×CV, about9×CV, about 8×CV, about 7×CV, or about 6×CV (inclusive); between about5×CV to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV,about 20×CV, about 18×CV, about 16×CV, about 14×CV, about 13×CV, about12×CV, about 11×CV, about 10×CV, about 9×CV, about 8×CV, or about 7×CV(inclusive); between about 6×CV to about 30×CV, about 28×CV, about26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV,about 14×CV, about 13×CV, about 12×CV, about 11×CV, about 10×CV, about9×CV, or about 8×CV (inclusive); between about 7×CV to about 30×CV,about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV, about18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV, about 11×CV,about 10×CV, or about 9×CV (inclusive); between about 8×CV to about30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV,about 18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV, about11×CV, or about 10×CV (inclusive); between about 9 CV to about 30×CV,about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV, about18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV, or about11×CV (inclusive); between about 10×CV to about 30×CV, about 28×CV,about 26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about16×CV, about 14×CV, about 13×CV, or about 12×CV (inclusive); betweenabout 11×CV to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about22×CV, about 20×CV, about 18×CV, about 16×CV, about 14×CV, or about13×CV (inclusive); between about 12×CV to about 30×CV, about 28×CV,about 26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about16×CV, or about 14×CV (inclusive); between about 14×CV to about 30×CV,about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV, about18×CV, or about 16×CV (inclusive); between about 16×CV to about 30×CV,about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV, orabout 18×CV (inclusive); between about 18×CV to about 30×CV, about28×CV, about 26×CV, about 24×CV, about 22×CV, or about 20×CV(inclusive); between about 20×CV to about 30×CV, about 28×CV, about26×CV, about 24×CV, or about 22×CV (inclusive); between about 22×CV toabout 30×CV, about 28×CV, about 26×CV, or about 24×CV (inclusive);between about 24×CV to about 30×CV, about 28×CV, or about 26×CV(inclusive); between about 26×CV to about 30×CV or about 28×CV(inclusive); or between about 28×CV to about 30×CV (inclusive). Thetotal time of the eluting can be, e.g., between about 2 minutes to about3 hours (e.g., between about 2 minutes to about 2.5 hours, between about2 minutes to about 2.0 hours, between about 2 minutes to about 1.5hours, between about 2 minutes to about 1.5 hours, between about 2minutes to about 1.25 hours, between about 2 minutes to about 1.25hours, between about 2 minutes to about 1 hour, between about 2 minutesand about 40 minutes, between about 10 minutes and about 40 minutes,between about 20 minutes and about 40 minutes, or between about 30minutes and 1.0 hour). Non-limiting examples of elution buffers that canbe used in these methods will depend on the resin and/or the therapeuticprotein. For example, an elution buffer can contain a differentconcentration of salt (e.g., increased salt concentration), a differentpH (e.g., an increased or decreased salt concentration), or a moleculethat will compete with the recombinant therapeutic protein for bindingto the resin. Examples of such elution buffers for each of the exemplarycapture mechanisms described herein are well known in the art.

Following the elution of the recombinant therapeutic protein from the atleast one chromatographic column or chromatographic membrane in thesecond MCCS that can be used to perform the unit operation of purifyingthe recombinant therapeutic protein, and before the next volume of fluidcontaining a recombinant therapeutic protein can be loaded onto the atleast one chromatographic column or chromatographic membrane, the atleast one chromatography column or chromatographic membrane must beequilibrated using an regeneration buffer. The regeneration buffer canbe passed through the at least one chromatography column orchromatographic membrane that can be used to perform the unit operationof purifying the recombinant therapeutic protein at a flow rate of,e.g., between about 0.2 mL/minute to about 100 L/minute (inclusive)(e.g., any of the subranges of this flow rate range described herein).The volume of regeneration buffer used to equilibrate the at least onechromatography column or chromatographic membrane that contains a resinthat can be used to perform the unit operation of purifying therecombinant therapeutic protein can be, e.g., between about 1× columnvolume (CV) to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about22×CV, about 20×CV, about 18×CV, about 16×CV, about 14×CV, about 13×CV,about 12×CV, about 11×CV, about 10×CV, about 9×CV, about 8×CV, about7×CV, about 6×CV, about 5×CV, about 4×CV, or about 3×CV (inclusive);between about 2×CV to about 30×CV, about 28×CV, about 26×CV, about24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV, about 14×CV,about 13×CV, about 12×CV, about 11×CV, about 10×CV, about 9×CV, about8×CV, about 7×CV, about 6×CV, about 5×CV, or about 4×CV (inclusive);between about 3×CV to about 30×CV, about 28×CV, about 26×CV, about24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV, about 14×CV,about 13×CV, about 12×CV, about 11×CV, about 10×CV, about 9×CV, about8×CV, about 7×CV, or about 5×CV (inclusive); between about 4×CV to about30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV,about 18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV, about11×CV, about 10×CV, about 9×CV, about 8×CV, about 7×CV, or about 6×CV(inclusive); between about 5×CV to about 30×CV, about 28×CV, about26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV,about 14×CV, about 13×CV, about 12×CV, about 11×CV, about 10×CV, about9×CV, about 8×CV, or about 7×CV (inclusive); between about 6×CV to about30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV,about 18×CV, about 16×CV, about 14×CV, about 13×CV, about 12×CV, about11×CV, about 10×CV, about 9×CV, or about 8×CV (inclusive); between about7×CV to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV,about 20×CV, about 18×CV, about 16×CV, about 14×CV, about 13×CV, about12×CV, about 11×CV, about 10×CV, or about 9×CV (inclusive); betweenabout 8×CV to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about22×CV, about 20×CV, about 18×CV, about 16×CV, about 14×CV, about 13×CV,about 12×CV, about 11×CV, or about 10×CV (inclusive); between about 9 CVto about 30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV,about 20×CV, about 18×CV, about 16×CV, about 14×CV, about 13×CV, about12×CV, or about 11×CV (inclusive); between about 10×CV to about 30×CV,about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV, about18×CV, about 16×CV, about 14×CV, about 13×CV, or about 12×CV(inclusive); between about 11×CV to about 30×CV, about 28×CV, about26×CV, about 24×CV, about 22×CV, about 20×CV, about 18×CV, about 16×CV,about 14×CV, or about 13×CV (inclusive); between about 12×CV to about30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, about 20×CV,about 18×CV, about 16×CV, or about 14×CV (inclusive); between about14×CV to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about22×CV, about 20×CV, about 18×CV, or about 16×CV (inclusive); betweenabout 16×CV to about 30×CV, about 28×CV, about 26×CV, about 24×CV, about22×CV, about 20×CV, or about 18×CV (inclusive); between about 18×CV toabout 30×CV, about 28×CV, about 26×CV, about 24×CV, about 22×CV, orabout 20×CV (inclusive); between about 20×CV to about 30×CV, about28×CV, about 26×CV, about 24×CV, or about 22×CV (inclusive); betweenabout 22×CV to about 30×CV, about 28×CV, about 26×CV, or about 24×CV(inclusive); between about 24×CV to about 30×CV, about 28×CV, or about26×CV (inclusive); between about 26×CV to about 30×CV or about 28×CV(inclusive); or between about 28×CV to about 30×CV (inclusive). Theconcentration of recombinant therapeutic protein in the eluate of the atleast one chromatography column or chromatographic membrane that can beused to perform the unit operation of purifying the recombinanttherapeutic protein can be, e.g., between about 0.05 mg/mL to about 100mg/mL recombinant therapeutic protein (e.g., between about 0.1 mg/mL toabout 90 mg/mL, between about 0.1 mg/mL to about 80 mg/mL, between about0.1 mg/mL to about 70 mg/mL, between about 0.1 mg/mL to about 60 mg/mL,between about 0.1 mg/mL to about 50 mg/mL, between about 0.1 mg/mL toabout 40 mg/mL, between about 2.5 mg/mL and about 7.5 mg/mL, betweenabout 0.1 mg/mL to about 30 mg/mL, between about 0.1 mg/mL to about 20mg/mL, between 0.5 mg/mL to about 20 mg/mL, between about 0.1 mg/mL toabout 15 mg/mL, between about 0.5 mg/mL to about 15 mg/mL, between about0.1 mg/mL to about 10 mg/mL, between about 0.5 mg/mL to about 10 mg/mL,between about 10 mg/mL to about 200 mg/mL, between about 10 mg/mL toabout 150 mg/mL, between about 10 mg/mL to about 100 mg/mL, betweenabout 10 mg/mL to about 50 mg/mL, between about 50 mg/mL to about 200mg/mL, between about 50 mg/mL to about 150 mg/mL, between about 50 mg/mLto about 100 mg/mL, between about 100 mg/mL to about 200 mg/mL, betweenabout 100 mg/mL to about 150 mg/mL, or about 150 mg/mL to about 200mg/mL recombinant therapeutic protein).

The at least one chromatography column or chromatographic membrane thatcan be used to perform the unit operation of polishing the recombinanttherapeutic protein can contain a resin that can be used to performcation exchange, anion exchange, or molecular sieve chromatography. Ascan be appreciated in the art, polishing a recombinant therapeuticprotein using the at least one chromatography column or chromatographymembrane that can be used to perform the unit operation of polishing therecombinant therapeutic protein in the second MCCS can include, e.g.,the steps of loading, chasing, and regenerating the at least onechromatography column or chromatographic membrane that can be used toperform the unit operation of polishing the recombinant therapeuticprotein. For example, when the steps of loading, chasing, andregenerating are used to perform the polishing, the recombinanttherapeutic protein does not bind the resin in the at least onechromatography column or chromatography membrane in the second MCCS thatis used to perform the unit operation of polishing the recombinanttherapeutic protein, and the recombinant therapeutic protein is elutedfrom the at least one chromatography column or chromatographic membranein the loading and chasing steps, and the regenerating step is used toremove any impurities from the at least one chromatography column orchromatographic membrane before additional fluid containing therecombinant therapeutic protein can be loaded onto the at least onechromatography column or chromatographic membrane. Exemplary flow ratesand buffer volumes to be used in each of the loading, chasing, andregenerating steps are described below.

The size, shape, and volume of the at least one chromatography column orchromatography membrane that can be used to perform the unit operationof polishing the recombinant therapeutic protein, and/or the size andshape of the at least one chromatographic membrane that can be used toperform the unit operation of polishing the recombinant therapeuticprotein can any of combination of the exemplary sizes, shapes, andvolumes of chromatography columns or chromatographic membranes describedherein. For example, the size of the at least one chromatography columnor chromatographic membrane that can be used to perform the unitoperation of polishing the recombinant therapeutic protein can have avolume of, e.g., between about 0.5 mL to about 200 L, about 180 L, about160 L, about 140 L, about 120 L, about 100 L, about 80 L, about 60 L,about 40 L, about 20 L, about 10 L, about 1 L, about 800 mL, about 600mL, about 400 mL, about 200 mL, about 180 mL, about 160 mL, about 140mL, about 120 mL, about 100 mL, about 80 mL, about 60 mL, about 40 mL,about 30 mL, about 20 mL, about 10 mL, about 5 mL, or about 2.5 mL(inclusive); between about 2.5 mL to about 200 L, about 180 L, about 160L, about 140 L, about 120 L, about 100 L, about 80 L, about 60 L, about40 L, about 20 L, about 10 L, about 1 L, about 800 mL, about 600 mL,about 400 mL, about 200 mL, about 180 mL, about 160 mL, about 140 mL,about 120 mL, about 100 mL, about 80 mL, about 60 mL, about 40 mL, about30 mL, about 20 mL, about 10 mL, or about 5 mL (inclusive); betweenabout 5 mL to about 200 L, about 180 L, about 160 L, about 140 L, about120 L, about 100 L, about 80 L, about 60 L, about 40 L, about 20 L,about 10 L, about 1 L, about 800 mL, about 600 mL, about 400 mL, about200 mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL, about100 mL, about 80 mL, about 60 mL, about 40 mL, about 30 mL, about 20 mL,or about 10 mL (inclusive); between about 10 mL to about 200 L, about180 L, about 160 L, about 140 L, about 120 L, about 100 L, about 80 L,about 60 L, about 40 L, about 20 L, about 10 L, about 1 L, about 800 mL,about 600 mL, about 400 mL, about 200 mL, about 180 mL, about 160 mL,about 140 mL, about 120 mL, about 100 mL, about 80 mL, about 60 mL,about 40 mL, about 30 mL, or about 20 mL (inclusive); between about 20mL to about 200 L, about 180 L, about 160 L, about 140 L, about 120 L,about 100 L, about 80 L, about 60 L, about 40 L, about 20 L, about 10 L,about 1 L, about 800 mL, about 600 mL, about 400 mL, about 200 mL, about180 mL, about 160 mL, about 140 mL, about 120 mL, about 100 mL, about 80mL, about 60 mL, about 40 mL, or about 30 mL (inclusive); between about30 mL to about 200 L, about 180 L, about 160 L, about 140 L, about 120L, about 100 L, about 80 L, about 60 L, about 40 L, about 20 L, about 10L, about 1 L, about 800 mL, about 600 mL, about 400 mL, about 200 mL,about 180 mL, about 160 mL, about 140 mL, about 120 mL, about 100 mL,about 80 mL, about 60 mL, or about 40 mL (inclusive); between about 40mL to about 200 L, about 180 L, about 160 L, about 140 L, about 120 L,about 100 L, about 80 L, about 60 L, about 40 L, about 20 L, about 10 L,about 1 L, about 800 mL, about 600 mL, about 400 mL, about 200 mL, about180 mL, about 160 mL, about 140 mL, about 120 mL, about 100 mL, about 80mL, or about 60 mL (inclusive); between about 60 mL to about 200 L,about 180 L, about 160 L, about 140 L, about 120 L, about 100 L, about80 L, about 60 L, about 40 L, about 20 L, about 10 L, about 1 L, about800 mL, about 600 mL, about 400 mL, about 200 mL, about 180 mL, about160 mL, about 140 mL, about 120 mL, about 100 mL, or about 80 mL(inclusive); between about 80 mL to about 200 L, about 180 L, about 160L, about 140 L, about 120 L, about 100 L, about 80 L, about 60 L, about40 L, about 20 L, about 10 L, about 1 L, about 800 mL, about 600 mL,about 400 mL, about 200 mL, about 180 mL, about 160 mL, about 140 mL,about 120 mL, or about 100 mL (inclusive); between about 100 mL to about200 L, about 180 L, about 160 L, about 140 L, about 120 L, about 100 L,about 80 L, about 60 L, about 40 L, about 20 L, about 10 L, about 1 L,about 800 mL, about 600 mL, about 400 mL, about 200 mL, about 180 mL,about 160 mL, or about 140 mL (inclusive); between about 140 mL to about200 L, about 180 L, about 160 L, about 140 L, about 120 L, about 100 L,about 80 L, about 60 L, about 40 L, about 20 L, about 10 L, about 1 L,about 800 mL, about 600 mL, about 400 mL, about 200 mL, about 180 mL, orabout 160 mL (inclusive); between about 160 mL to about 200 L, about 180L, about 160 L, about 140 L, about 120 L, about 100 L, about 80 L, about60 L, about 40 L, about 20 L, about 10 L, about 1 L, about 800 mL, about600 mL, about 400 mL, about 200 mL, or about 180 mL (inclusive); betweenabout 180 mL to about 200 L, about 180 L, about 160 L, about 140 L,about 120 L, about 100 L, about 80 L, about 60 L, about 40 L, about 20L, about 10 L, about 1 L, about 800 mL, about 600 mL, about 400 mL, orabout 200 mL (inclusive); between about 200 mL to about 200 L, about 180L, about 160 L, about 140 L, about 120 L, about 100 L, about 80 L, about60 L, about 40 L, about 20 L, about 10 L, about 1 L, about 800 mL, about600 mL, or about 400 mL (inclusive); between about 400 mL to about 200L, about 180 L, about 160 L, about 140 L, about 120 L, about 100 L,about 80 L, about 60 L, about 40 L, about 20 L, about 10 L, about 1 L,about 800 mL, or about 600 mL (inclusive); between about 600 mL to about200 L, about 180 L, about 160 L, about 140 L, about 120 L, about 100 L,about 80 L, about 60 L, about 40 L, about 20 L, about 10 L, about 1 L,or about 800 mL (inclusive); between about 800 mL to about 200 L, about180 L, about 160 L, about 140 L, about 120 L, about 100 L, about 80 L,about 60 L, about 40 L, about 20 L, about 10 L, or about 1 L(inclusive); between about 1 L to about 200 L, about 180 L, about 160 L,about 140 L, about 120 L, about 100 L, about 80 L, about 60 L, about 40L, about 20 L, or about 10 L (inclusive); between about 10 L to about200 L, about 180 L, about 160 L, about 140 L, about 120 L, about 100 L,about 80 L, about 60 L, about 40 L, or about 20 L (inclusive); betweenabout 20 L to about 200 L, about 180 L, about 160 L, about 140 L, about120 L, about 100 L, about 80 L, about 60 L, or about 40 L (inclusive);between about 40 L to about 200 L, about 180 L, about 160 L, about 140L, about 120 L, about 100 L, about 80 L, or about 60 L (inclusive);between about 60 L to about 200 L, about 180 L, about 160 L, about 140L, about 120 L, about 100 L, or about 80 L (inclusive); between about 80L to about 200 L, about 180 L, about 160 L, about 140 L, about 120 L, orabout 100 L (inclusive); between about 100 L to about 200 L, about 180L, about 160 L, about 140 L, or about 120 L (inclusive); between about120 L to about 200 L, about 180 L, about 160 L, or about 140 L(inclusive); between about 140 L to about 200 L, about 180 L, or about160 L (inclusive); between about 160 L to about 200 L or about 180 L(inclusive); or between about 180 L to about 200 L (inclusive). The flowrate of the fluid containing the recombinant therapeutic protein as itis loaded onto the at least one chromatography column or chromatographicmembrane that can be used to perform the unit operation of polishing therecombinant therapeutic protein can be, e.g., between about 0.1mL/minute to about 100 L/minute, about 80 L/minute, about 60 L/minute,about 40 L/minute, about 20 L/minute, about 10 L/minute, about 5L/minute, about 1 L/minute, about 800 mL/minute, about 600 mL/minute,about 400 mL/minute, about 200 mL/minute, about 100 mL/minute, about 50mL/minute, about 25 mL/minute, about 12.5 mL/minute, about 10.0mL/minute, about 8.0 mL/minute, about 6 mL/minute, about 4 mL/minute,about 3 mL/minute, about 2 mL/minute, about 1 mL/minute, about 0.5mL/minute, or about 0.2 mL/minute (inclusive); between about 0.2mL/minute to about 100 L/minute, about 80 L/minute, about 60 L/minute,about 40 L/minute, about 20 L/minute, about 10 L/minute, about 5L/minute, about 1 L/minute, about 800 mL/minute, about 600 mL/minute,about 400 mL/minute, about 200 mL/minute, about 100 mL/minute, about 50mL/minute, about 25 mL/minute, about 12.5 mL/minute, about 10.0mL/minute, about 8.0 mL/minute, about 6 mL/minute, about 4 mL/minute,about 3 mL/minute, about 2 mL/minute, or about 1 mL/minute (inclusive);between about 1 mL/minute to about 100 L/minute, about 80 L/minute,about 60 L/minute, about 40 L/minute, about 20 L/minute, about 10L/minute, about 5 L/minute, about 1 L/minute, about 800 mL/minute, about600 mL/minute, about 400 mL/minute, about 200 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, about 12.5 mL/minute,about 10.0 mL/minute, about 8.0 mL/minute, about 6 mL/minute, about 4mL/minute, about 3 mL/minute, or about 2 mL/minute (inclusive); betweenabout 2 mL/minute to about 100 L/minute, about 80 L/minute, about 60L/minute, about 40 L/minute, about 20 L/minute, about 10 L/minute, about5 L/minute, about 1 L/minute, about 800 mL/minute, about 600 mL/minute,about 400 mL/minute, about 200 mL/minute, about 100 mL/minute, about 50mL/minute, about 25 mL/minute, about 12.5 mL/minute, about 10.0mL/minute, about 8.0 mL/minute, about 6 mL/minute, about 4 mL/minute, orabout 3 mL/minute (inclusive); between about 3 mL/minute to about 100L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute, about20 L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute,about 800 mL/minute, about 600 mL/minute, about 400 mL/minute, about 200mL/minute, about 100 mL/minute, about 50 mL/minute, about 25 mL/minute,about 12.5 mL/minute, about 10.0 mL/minute, about 8.0 mL/minute, about 6mL/minute, or about 4 mL/minute (inclusive); between about 4 mL/minuteto about 100 L/minute, about 80 L/minute, about 60 L/minute, about 40L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 800 mL/minute, about 600 mL/minute, about 400mL/minute, about 200 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute, about 12.5 mL/minute, about 10.0 mL/minute, about8.0 mL/minute, or about 6 mL/minute (inclusive); between about 6mL/minute to about 100 L/minute, about 80 L/minute, about 60 L/minute,about 40 L/minute, about 20 L/minute, about 10 L/minute, about 5L/minute, about 1 L/minute, about 800 mL/minute, about 600 mL/minute,about 400 mL/minute, about 200 mL/minute, about 100 mL/minute, about 50mL/minute, about 25 mL/minute, about 12.5 mL/minute, about 10.0mL/minute, or about 8.0 mL/minute (inclusive); between about 8 mL/minuteto about 100 L/minute, about 80 L/minute, about 60 L/minute, about 40L/minute, about 20 L/minute, about 10 L/minute, about 5 L/minute, about1 L/minute, about 800 mL/minute, about 600 mL/minute, about 400mL/minute, about 200 mL/minute, about 100 mL/minute, about 50 mL/minute,about 25 mL/minute, about 12.5 mL/minute, or about 10.0 mL/minute(inclusive); between about 10 mL/minute to about 100 L/minute, about 80L/minute, about 60 L/minute, about 40 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 800 mL/minute,about 600 mL/minute, about 400 mL/minute, about 200 mL/minute, about 100mL/minute, about 50 mL/minute, about 25 mL/minute, or about 12.5mL/minute (inclusive); between about 12.5 mL/minute to about 100L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute, about20 L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute,about 800 mL/minute, about 600 mL/minute, about 400 mL/minute, about 200mL/minute, about 100 mL/minute, about 50 mL/minute, or about 25mL/minute (inclusive); between about 25 mL/minute to about 100 L/minute,about 80 L/minute, about 60 L/minute, about 40 L/minute, about 20L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute, about800 mL/minute, about 600 mL/minute, about 400 mL/minute, about 200mL/minute, about 100 mL/minute, or about 50 mL/minute (inclusive);between about 50 mL/minute to about 100 L/minute, about 80 L/minute,about 60 L/minute, about 40 L/minute, about 20 L/minute, about 10L/minute, about 5 L/minute, about 1 L/minute, about 800 mL/minute, about600 mL/minute, about 400 mL/minute, about 200 mL/minute, or about 100mL/minute (inclusive); between about 100 mL/minute to about 100L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute, about20 L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute,about 800 mL/minute, about 600 mL/minute, about 400 mL/minute, or about200 mL/minute (inclusive); between about 200 mL/minute to about 100L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute, about20 L/minute, about 10 L/minute, about 5 L/minute, about 1 L/minute,about 800 mL/minute, about 600 mL/minute, or about 400 mL/minute(inclusive); between about 400 mL/minute to about 100 L/minute, about 80L/minute, about 60 L/minute, about 40 L/minute, about 20 L/minute, about10 L/minute, about 5 L/minute, about 1 L/minute, about 800 mL/minute, orabout 600 mL/minute (inclusive); between about 600 mL/minute to about100 L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute,about 20 L/minute, about 10 L/minute, about 5 L/minute, about 1L/minute, or about 800 mL/minute (inclusive); between about 800mL/minute to about 100 L/minute, about 80 L/minute, about 60 L/minute,about 40 L/minute, about 20 L/minute, about 10 L/minute, about 5L/minute, or about 1 L/minute (inclusive); between about 1 L/minute toabout 100 L/minute, about 80 L/minute, about 60 L/minute, about 40L/minute, about 20 L/minute, about 10 L/minute, or about 5 L/minute(inclusive); between about 5 L/minute to about 100 L/minute, about 80L/minute, about 60 L/minute, about 40 L/minute, about 20 L/minute, orabout 10 L/minute (inclusive); between about 10 L/minute to about 100L/minute, about 80 L/minute, about 60 L/minute, about 40 L/minute, orabout 20 L/minute (inclusive); between about 20 L/minute to about 100L/minute, about 80 L/minute, about 60 L/minute, or about 40 L/minute(inclusive); between about 40 L/minute to about 100 L/minute, about 80L/minute, or about 60 L/minute (inclusive); between about 60 L/minute toabout 100 L/minute or about 80 L/minute (inclusive); or between about 80L/minute to about 100 L/minute (inclusive). The total volume of fluidcontaining a recombinant therapeutic protein loaded onto the at leastone chromatography column or chromatographic membrane that can be usedto perform the unit operation of polishing the recombinant therapeuticprotein can be, e.g., between about 1.0 mL to about 1,000 L, about 900L, about 800 L, about 700 L, about 600 L, about 500 L, about 400 L,about 300 L, about 200 L, about 100 L, about 50 L, about 25 L, about 10L, about 5 L, about 1 L, about 800 mL, about 600 mL, about 400 mL, about250 mL, about 225 mL, about 200 mL, about 175 mL, about 150 mL, about125 mL, about 100 mL, about 75 mL, about 50 mL, about 25 mL, about 10mL, or about 5 mL (inclusive); between about 5 mL to about 1,000 L,about 900 L, about 800 L, about 700 L, about 600 L, about 500 L, about400 L, about 300 L, about 200 L, about 100 L, about 50 L, about 25 L,about 10 L, about 5 L, about 1 L, about 800 mL, about 600 mL, about 400mL, about 250 mL, about 225 mL, about 200 mL, about 175 mL, about 150mL, about 125 mL, about 100 mL, about 75 mL, about 50 mL, about 25 mL,or about 10 mL (inclusive); between about 10 mL to about 1,000 L, about900 L, about 800 L, about 700 L, about 600 L, about 500 L, about 400 L,about 300 L, about 200 L, about 100 L, about 50 L, about 25 L, about 10L, about 5 L, about 1 L, about 800 mL, about 600 mL, about 400 mL, about250 mL, about 225 mL, about 200 mL, about 175 mL, about 150 mL, about125 mL, about 100 mL, about 75 mL, about 50 mL, or about 25 mL(inclusive); between about 25 mL to about 1,000 L, about 900 L, about800 L, about 700 L, about 600 L, about 500 L, about 400 L, about 300 L,about 200 L, about 100 L, about 50 L, about 25 L, about 10 L, about 5 L,about 1 L, about 800 mL, about 600 mL, about 400 mL, about 250 mL, about225 mL, about 200 mL, about 175 mL, about 150 mL, about 125 mL, about100 mL, about 75 mL, or about 50 mL (inclusive); between about 50 mL toabout 1,000 L, about 900 L, about 800 L, about 700 L, about 600 L, about500 L, about 400 L, about 300 L, about 200 L, about 100 L, about 50 L,about 25 L, about 10 L, about 5 L, about 1 L, about 800 mL, about 600mL, about 400 mL, about 250 mL, about 225 mL, about 200 mL, about 175mL, about 150 mL, about 125 mL, about 100 mL, or about 75 mL(inclusive); between about 75 mL to about 1,000 L, about 900 L, about800 L, about 700 L, about 600 L, about 500 L, about 400 L, about 300 L,about 200 L, about 100 L, about 50 L, about 25 L, about 10 L, about 5 L,about 1 L, about 800 mL, about 600 mL, about 400 mL, about 250 mL, about225 mL, about 200 mL, about 175 mL, about 150 mL, about 125 mL, or about100 mL (inclusive); between about 100 mL to about 1,000 L, about 900 L,about 800 L, about 700 L, about 600 L, about 500 L, about 400 L, about300 L, about 200 L, about 100 L, about 50 L, about 25 L, about 10 L,about 5 L, about 1 L, about 800 mL, about 600 mL, about 400 mL, about250 mL, about 225 mL, about 200 mL, about 175 mL, about 150 mL, or about125 mL (inclusive); between about 125 mL to about 1,000 L, about 900 L,about 800 L, about 700 L, about 600 L, about 500 L, about 400 L, about300 L, about 200 L, about 100 L, about 50 L, about 25 L, about 10 L,about 5 L, about 1 L, about 800 mL, about 600 mL, about 400 mL, about250 mL, about 225 mL, about 200 mL, about 175 mL, or about 150 mL(inclusive); between about 150 mL to about 1,000 L, about 900 L, about800 L, about 700 L, about 600 L, about 500 L, about 400 L, about 300 L,about 200 L, about 100 L, about 50 L, about 25 L, about 10 L, about 5 L,about 1 L, about 800 mL, about 600 mL, about 400 mL, about 250 mL, about225 mL, about 200 mL, or about 175 mL (inclusive); between about 175 mLto about 1,000 L, about 900 L, about 800 L, about 700 L, about 600 L,about 500 L, about 400 L, about 300 L, about 200 L, about 100 L, about50 L, about 25 L, about 10 L, about 5 L, about 1 L, about 800 mL, about600 mL, about 400 mL, about 250 mL, about 225 mL, or about 200 mL(inclusive); between about 200 mL to about 1,000 L, about 900 L, about800 L, about 700 L, about 600 L, about 500 L, about 400 L, about 300 L,about 200 L, about 100 L, about 50 L, about 25 L, about 10 L, about 5 L,about 1 L, about 800 mL, about 600 mL, about 400 mL, about 250 mL, orabout 225 mL (inclusive); between about 225 mL to about 1,000 L, about900 L, about 800 L, about 700 L, about 600 L, about 500 L, about 400 L,about 300 L, about 200 L, about 100 L, about 50 L, about 25 L, about 10L, about 5 L, about 1 L, about 800 mL, about 600 mL, about 400 mL, orabout 250 mL (inclusive); between about 250 mL to about 1,000 L, about900 L, about 800 L, about 700 L, about 600 L, about 500 L, about 400 L,about 300 L, about 200 L, about 100 L, about 50 L, about 25 L, about 10L, about 5 L, about 1 L, about 800 mL, about 600 mL, or about 400 mL(inclusive); between about 400 mL to about 1,000 L, about 900 L, about800 L, about 700 L, about 600 L, about 500 L, about 400 L, about 300 L,about 200 L, about 100 L, about 50 L, about 25 L, about 10 L, about 5 L,about 1 L, about 800 mL, or about 600 mL (inclusive); between about 600mL to about 1,000 L, about 900 L, about 800 L, about 700 L, about 600 L,about 500 L, about 400 L, about 300 L, about 200 L, about 100 L, about50 L, about 25 L, about 10 L, about 5 L, about 1 L, or about 800 mL(inclusive); between about 800 mL to about 1,000 L, about 900 L, about800 L, about 700 L, about 600 L, about 500 L, about 400 L, about 300 L,about 200 L, about 100 L, about 50 L, about 25 L, about 10 L, about 5 L,or about 1 L (inclusive); between about 1 L to about 1,000 L, about 900L, about 800 L, about 700 L, about 600 L, about 500 L, about 400 L,about 300 L, about 200 L, about 100 L, about 50 L, about 25 L, about 10L, or about 5 L (inclusive); between about 5 L to about 1,000 L, about900 L, about 800 L, about 700 L, about 600 L, about 500 L, about 400 L,about 300 L, about 200 L, about 100 L, about 50 L, about 25 L, or about10 L (inclusive); between about 10 L to about 1,000 L, about 900 L,about 800 L, about 700 L, about 600 L, about 500 L, about 400 L, about300 L, about 200 L, about 100 L, about 50 L, or about 25 L (inclusive);between about 25 L to about 1,000 L, about 900 L, about 800 L, about 700L, about 600 L, about 500 L, about 400 L, about 300 L, about 200 L,about 100 L, or about 50 L (inclusive); between about 50 L to about1,000 L, about 900 L, about 800 L, about 700 L, about 600 L, about 500L, about 400 L, about 300 L, about 200 L, or about 100 L (inclusive);between about 100 L to about 1,000 L, about 900 L, about 800 L, about700 L, about 600 L, about 500 L, about 400 L, about 300 L, or about 200L (inclusive); between about 200 L to about 1,000 L, about 900 L, about800 L, about 700 L, about 600 L, about 500 L, or about 400 L(inclusive); between about 400 L to about 1,000 L, about 900 L, about800 L, about 700 L, about 600 L, or about 500 L (inclusive); betweenabout 500 L to about 1,000 L, about 900 L, about 800 L, about 700 L, orabout 600 L (inclusive); between about 600 L to about 1,000 L, about 900L, about 800 L, or about 700 L (inclusive); between about 700 L to about1,000 L, about 900 L, or about 800 L (inclusive); between about 800 L toabout 1,000 L or about 900 L (inclusive); or about 900 L to about 1,000L (inclusive). The resin in the at least one chromatography column orchromatographic membrane used to perform the polishing can be an anionexchange or cation exchange resin. The resin in the at least onechromatography column or chromatographic membrane that is used toperform the unit operation of polishing can be a cationic exchange resin(e.g., Sartobind® Q resin, Sartorius, Goettingen, Germany).

Following the loading step, the at least one chromatographic column orchromatographic membrane in the second MCCS that can be used to performthe unit operation of polishing the recombinant therapeutic protein, achasing step is performed (e.g., a chase buffer is passed through the atleast one chromatography membrane or chromatographic membrane to collectthe recombinant therapeutic protein which does not substantially bind tothe at least one chromatography column or chromatographic membrane). Inthese examples, the chase buffer can be passed through the at least onechromatography column or chromatographic membrane at a flow rate of,e.g., between about 0.2 mL/minute to about 100 L/minute (inclusive)(e.g., any of the subranges of this flow rate range described herein).The volume of chase buffer used can be, e.g., between about 1× columnvolume (CV) to about 100×CV (e.g., between about 1×CV to about 90×CV,between about 1×CV to about 80×CV, between about 1×CV to about 70×CV,between about 1×CV to about 60×CV, between about 1× to about 50×CV,between about 1×CV to about 40×CV, between about 1×CV to about 30×CV,between about 1×CV to about 20×CV, between about 1×CV to about 15×CV,between about 5×CV to about 20×CV, between about 5×CV to about 30×CV,between about 1×CV to about 14×CV, about 1×CV to about 13×CV, about 1×CVto about 12×CV, about 1×CV to about 11×CV, about 2×CV to about 11×CV,about 3×CV to about 11×CV, about 4×CV to about 11×CV, about 2.5×CV toabout 5.0×CV, about 5×CV to about 11×CV, or about 5×CV to about 10×CV).The total time of the chasing can be, e.g., between about 1 minute toabout 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16hours, about 14 hours, about 12 hours, about 10 hours, about 8 hours,about 6 hours, about 4 hours, about 3 hours, about 2 hours, about 1.5hours, about 1.25 hours, about 1 hour, about 30 minutes, about 20minutes, about 10 minutes, about 5 minutes, or about 2 minutes(inclusive); between about 2 minutes to about 24 hours, about 22 hours,about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours,about 3 hours, about 2 hours, about 1.5 hours, about 1.25 hours, about 1hour, about 30 minutes, about 20 minutes, about 10 minutes, or about 5minutes (inclusive); between about 5 minutes to about 24 hours, about 22hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours,about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4hours, about 3 hours, about 2 hours, about 1.5 hours, about 1.25 hours,about 1 hour, about 30 minutes, about 20 minutes, or about 10 minutes(inclusive); between about 10 minutes to about 24 hours, about 22 hours,about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours,about 3 hours, about 2 hours, about 1.5 hours, about 1.25 hours, about 1hour, about 30 minutes, or about 20 minutes (inclusive); between about20 minutes to about 24 hours, about 22 hours, about 20 hours, about 18hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours,about 8 hours, about 6 hours, about 4 hours, about 3 hours, about 2hours, about 1.5 hours, about 1.25 hours, about 1 hour, or about 30minutes (inclusive); between about 30 minutes to about 24 hours, about22 hours, about 20 hours, about 18 hours, about 16 hours, about 14hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours,about 4 hours, about 3 hours, about 2 hours, about 1.5 hours, about 1.25hours, or about 1 hour (inclusive); between about 1 hour to about 24hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours,about 14 hours, about 12 hours, about 10 hours, about 8 hours, about 6hours, about 4 hours, about 3 hours, about 2 hours, about 1.5 hours, orabout 1.25 hours (inclusive); between about 1.25 hours to about 24hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours,about 14 hours, about 12 hours, about 10 hours, about 8 hours, about 6hours, about 4 hours, about 3 hours, about 2 hours, or about 1.5 hours(inclusive); between about 1.5 hours to about 24 hours, about 22 hours,about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours,about 3 hours, or about 2 hours (inclusive); between about 2 hours toabout 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16hours, about 14 hours, about 12 hours, about 10 hours, about 8 hours,about 6 hours, about 4 hours, or about 3 hours (inclusive); betweenabout 3 hours to about 24 hours, about 22 hours, about 20 hours, about18 hours, about 16 hours, about 14 hours, about 12 hours, about 10hours, about 8 hours, about 6 hours, or about 4 hours (inclusive);between about 4 hours to about 24 hours, about 22 hours, about 20 hours,about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10hours, about 8 hours, or about 6 hours (inclusive); between about 6hours to about 24 hours, about 22 hours, about 20 hours, about 18 hours,about 16 hours, about 14 hours, about 12 hours, about 10 hours, or about8 hours (inclusive); between about 8 hours to about 24 hours, about 22hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours,about 12 hours, or about 10 hours (inclusive); between about 10 hours toabout 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16hours, about 14 hours, or about 12 hours (inclusive); between about 12hours to about 24 hours, about 22 hours, about 20 hours, about 18 hours,about 16 hours, or about 14 hours (inclusive); between about 14 hours toabout 24 hours, about 22 hours, about 20 hours, about 18 hours, or about16 hours (inclusive); between about 16 hours to about 24 hours, about 22hours, about 20 hours, or about 18 hours (inclusive); between about 18hours to about 24 hours, about 22 hours, or about 20 hours (inclusive);between about 20 hours to about 24 hours to about 22 hours (inclusive);or between about 22 hours to about 24 hours (inclusive). The combinedconcentration of therapeutic recombinant protein present in the eluatecoming through the column in the loading step and the chasing step canbe, e.g., between about 0.1 mg/mL to about 100 mg/mL recombinant protein(e.g., between about 0.1 mg/mL to about 90 mg/mL, between about 0.1mg/mL to about 80 mg/mL, between about 0.1 mg/mL to about 70 mg/mL,between about 0.1 mg/mL to about 60 mg/mL, between about 0.1 mg/mL toabout 50 mg/mL, between about 0.1 mg/mL to about 40 mg/mL, between about2.5 mg/mL and about 7.5 mg/mL, between about 0.1 mg/mL to about 30mg/mL, between about 0.1 mg/mL to about 20 mg/mL, between 0.5 mg/mL toabout 20 mg/mL, between about 0.1 mg/mL to about 15 mg/mL, between about0.5 mg/mL to about 15 mg/mL, between about 0.1 mg/mL to about 10 mg/mL,between about 0.5 mg/mL to about 10 mg/mL, or between about 1 mg/mL andabout 5 mg/mL recombinant therapeutic protein).

Following the chasing step and before the next volume fluid containing arecombinant therapeutic protein can be loaded onto the at least onechromatographic column or chromatographic membrane that can be used toperform the unit operation of polishing, the at least one chromatographycolumn or chromatographic membrane must be regenerated using aregeneration buffer. The regeneration buffer can be passed through theat least one chromatography column or chromatographic membrane that canbe used to perform the unit operation of polishing the recombinanttherapeutic protein at a flow rate of, e.g., between about 0.2 mL/minuteto about 100 L/minute (inclusive) (e.g., any of the subranges of thisflow rate range described herein). The volume of regeneration bufferused to regenerate the at least one chromatography column orchromatographic membrane that can be used to perform the unit operationof polishing can be, e.g., between about 1× column volume (CV) to about500×CV (e.g., between about 1×CV to about 450×CV, between about 1×CV toabout 400×CV, between about 1×CV to about 350×CV, between about 1×CV toabout 300×CV, between about 1×CV to about 250×CV, between about 1×CV toabout 200×CV, between about 1×CV to about 150×CV, between about 1×CV toabout 100×CV, between about 1×CV to about 90×CV, between about 1×CV toabout 80×CV, between about 1×CV to about 70×CV, between about 1×CV toabout 60×CV, between about 1× to about 50×CV, between about 1×CV toabout 40×CV, between about 1×CV to about 30×CV, between about 1×CV toabout 20×CV, between about 1×CV to about 15×CV, between about 5×CV toabout 20×CV, between about 5×CV to about 30×CV, between about 1×CV toabout 14×CV, about 1×CV to about 13×CV, about 1×CV to about 12×CV, about1×CV to about 11×CV, about 2×CV to about 11×CV, about 3×CV to about11×CV, about 4×CV to about 11×CV, about 2.5×CV to about 5.0×CV, about5×CV to about 11×CV, or about 5×CV to about 10×CV).

In other examples, the one or more chromatography column(s) and/orchromatographic membranes used to perform the unit operation ofpolishing contain a resin that selectively binds or retains theimpurities present in a fluid containing the recombinant therapeuticprotein, and instead of regenerating the one or more column(s) and/ormembrane(s), the one or more column(s) and/or membrane(s) are replaced(e.g., replaced with a substantially similar column(s) and/ormembrane(s)) once the binding capacity of the resin in the one or morecolumn(s) and/or membrane(s) has been reached or is substantially closeto being reached.

In some examples of these processes, the second MCCS includes a PCCScontaining three chromatography columns and one chromatographicmembrane, e.g., where the three chromatography columns in the PCCSperform the unit operation of purifying the recombinant therapeuticprotein (e.g., using at least one chromatography column(s) that can beused to perform the unit of operation of purifying the protein) and thechromatographic membrane in the PCCS performs the unit operation ofpolishing the recombinant therapeutic protein. In these examples, thechromatographic membrane in the PCCS that can be used to perform theunit operation of polishing the recombinant therapeutic protein can beany of the exemplary chromatographic membranes described herein that canbe used to perform the unit operation of polishing the recombinanttherapeutic protein. Any of the column switching methods describedherein can be used to determine when the first three chromatographycolumns and the chromatographic membrane in the PCCS in this example canbe switched.

Some embodiments of this example can further include a step of adjustingthe ionic concentration and/or pH of the eluate from the threechromatographic columns in the PCCS before the eluate is fed into thechromatographic membrane in the PCCS. As described herein, the ionicconcentration and/or pH of the eluate from the three chromatographycolumns in PCCS can be adjusted (before it is fed into thechromatographic membrane in the PCCS in this example)) by adding abuffer to the eluate of the three chromatography columns in the PCCS(e.g., through the use of an in-line buffer adjustment reservoir). Thebuffer can be added to the eluate at a flow rate of, e.g., between about0.2 mL/minute to about 100 L/minute (inclusive) (e.g., any of thesubranges of this flow rate range described herein).

These examples can further include a step of holding or storing theeluate from the three chromatography columns in the PCCS in this exampleprior to feeding the eluate into the chromatographic membrane(chromatographic membrane that can be used to perform the unit operationof polishing the recombinant therapeutic protein). As described herein,this holding or storing step can be performed using any of thereservoirs (e.g., back-up tanks) described herein.

These examples can also include a step of filtering the eluate from thechromatographic membrane in the exemplary PCCS system (eluate of thechromatographic membrane that can be used to perform the unit operationof polishing the recombinant therapeutic protein). Any of the exemplaryfilters or methods for filtration described herein can be used to filterthe eluate from the chromatographic membrane in this exemplary PCCS(eluate of the chromatographic membrane that can be used to perform theunit operation of polishing the recombinant therapeutic protein).

As can be appreciated by those in the art, the therapeutic protein drugsubstance can be periodically eluted from the second MCCS using any ofthe processes described herein. For example, any of the processesdescribed herein can elute the therapeutic protein drug substance for aduration of, e.g., between about 30 seconds and about 48 hours, about 44hours, about 40 hours, about 36 hours, about 32 hours, about 28 hours,about 24 hours, about 20 hours, about 16 hours, about 12 hours, about 8hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about1.5 hours, about 1 hour, about 30 minutes, about 20 minutes, about 10minutes, about 5 minutes, or about 1 minute (inclusive); between about 1minute and about 48 hours, about 44 hours, about 40 hours, about 36hours, about 32 hours, about 28 hours, about 24 hours, about 20 hours,about 16 hours, about 12 hours, about 8 hours, about 5 hours, about 4hours, about 3 hours, about 2 hours, about 1.5 hours, about 1 hour,about 30 minutes, about 20 minutes, about 10 minutes, and about 5minutes (inclusive); between about 5 minutes and about 48 hours, about44 hours, about 40 hours, about 36 hours, about 32 hours, about 28hours, about 24 hours, about 20 hours, about 16 hours, about 12 hours,about 8 hours, about 5 hours, about 4 hours, about 3 hours, about 2hours, about 1.5 hours, about 1 hour, about 30 minutes, about 20minutes, or about 10 minutes (inclusive); between about 10 minutes andabout 48 hours, about 44 hours, about 40 hours, about 36 hours, about 32hours, about 28 hours, about 24 hours, about 20 hours, about 16 hours,about 12 hours, about 8 hours, about 5 hours, about 4 hours, about 3hours, about 2 hours, about 1.5 hours, about 1 hour, about 30 minutes,or about 20 minutes (inclusive); between about 20 minutes and about 48hours, about 44 hours, about 40 hours, about 36 hours, about 32 hours,about 28 hours, about 24 hours, about 20 hours, about 16 hours, about 12hours, about 8 hours, about 5 hours, about 4 hours, about 3 hours, about2 hours, about 1.5 hours, about 1 hour, or about 30 minutes (inclusive);between about 30 minutes and about 48 hours, about 44 hours, about 40hours, about 36 hours, about 32 hours, about 28 hours, about 24 hours,about 20 hours, about 16 hours, about 12 hours, about 8 hours, about 5hours, about 4 hours, about 3 hours, about 2 hours, about 1.5 hours, orabout 1 hour (inclusive); between about 1 hour and about 48 hours, about44 hours, about 40 hours, about 36 hours, about 32 hours, about 28hours, about 24 hours, about 20 hours, about 16 hours, about 12 hours,about 8 hours, about 5 hours, about 4 hours, about 3 hours, about 2hours, or about 1.5 hours (inclusive); between about 1.5 hours and about48 hours, about 44 hours, about 40 hours, about 36 hours, about 32hours, about 28 hours, about 24 hours, about 20 hours, about 16 hours,about 12 hours, about 8 hours, about 5 hours, about 4 hours, about 3hours, or about 2 hours (inclusive); between about 2 hours and about 48hours, about 44 hours, about 40 hours, about 36 hours, about 32 hours,about 28 hours, about 24 hours, about 20 hours, about 16 hours, about 12hours, about 8 hours, about 5 hours, about 4 hours, or about 3 hours(inclusive); between about 3 hours and about 48 hours, about 44 hours,about 40 hours, about 36 hours, about 32 hours, about 28 hours, about 24hours, about 20 hours, about 16 hours, about 12 hours, about 8 hours,about 5 hours, or about 4 hours (inclusive); between about 4 hours andabout 48 hours, about 44 hours, about 40 hours, about 36 hours, about 32hours, about 28 hours, about 24 hours, about 20 hours, about 16 hours,about 12 hours, about 8 hours, or about 5 hours (inclusive); betweenabout 5 hours and about 48 hours, about 44 hours, about 40 hours, about36 hours, about 32 hours, about 28 hours, about 24 hours, about 20hours, about 16 hours, about 12 hours, or about 8 hours (inclusive);between about 8 hours and about 48 hours, about 44 hours, about 40hours, about 36 hours, about 32 hours, about 28 hours, about 24 hours,about 20 hours, about 16 hours, or about 12 hours (inclusive); betweenabout 12 hours and about 48 hours, about 44 hours, about 40 hours, about36 hours, about 32 hours, about 28 hours, about 24 hours, about 20hours, or about 16 hours (inclusive); between about 16 hours and about48 hours, about 44 hours, about 40 hours, about 36 hours, about 32hours, about 28 hours, about 24 hours, or about 20 hours (inclusive);between about 20 hours and about 48 hours, about 44 hours, about 40hours, about 36 hours, about 32 hours, about 28 hours, or about 24 hours(inclusive); between about 24 hours and about 48 hours, about 44 hours,about 40 hours, about 36 hours, about 32 hours, or about 28 hours(inclusive); between about 28 hours and about 48 hours, about 44 hours,about 40 hours, about 36 hours, or about 32 hours (inclusive); betweenabout 32 hours and about 48 hours, about 44 hours, about 40 hours, orabout 36 hours (inclusive); between about 36 hours and about 48 hours,about 44 hours, or about 40 hours (inclusive); between about 40 hoursand about 48 hours or about 44 hours (inclusive); or between about 44hours and about 48 hours (inclusive), depending on, e.g., thechromatography column(s) and/or chromatographic membrane(s) used in thefirst and second MCCS.

Culturing Methods

Some of the processes described herein further include a step ofculturing cells (e.g., recombinant mammalian cells) that secrete arecombinant therapeutic protein in a bioreactor (e.g., a perfusion orfed-batch bioreactor) that contains a liquid culture medium, wherein avolume of the liquid culture medium that is substantially free of cells(e.g., mammalian cells) is continuously or periodically removed from theperfusion bioreactor and fed into the first multi-column chromatographysystem (MCC1). The bioreactor can have a volume of, e.g., between about1 L to about 10,000 L (e.g., between about 1 L to about 50 L, betweenabout 50 L to about 500 L, between about 500 L to about 1000 L, between500 L to about 5000 L, between about 500 L to about 10,000 L, betweenabout 5000 L to about 10,000 L, between about 1 L and about 10,000 L,between about 1 L and about 8,000 L, between about 1 L and about 6,000L, between about 1 L and about 5,000 L, between about 100 L and about5,000 L, between about 10 L and about 100 L, between about 10 L andabout 4,000 L, between about 10 L and about 3,000 L, between about 10 Land about 2,000 L, or between about 10 L and about 1,000 L). The amountof liquid culture medium present in a bioreactor can be, e.g., betweenabout between about 0.5 L to about 5,000 L (e.g., between about 0.5 L toabout 25 L, between about 25 L to about 250 L, between about 250 L toabout 500 L, between 250 L to about 2500 L, between about 250 L to about5,000 L, between about 2500 L to about 5,000 L, between about 0.5 L andabout 5,000 L, between about 0.5 L and about 4,000 L, between about 0.5L and about 3,000 L, between about 0.5 L and about 2,500 L, betweenabout 50 L and about 2,500 L, between about 5 L and about 50 L, betweenabout 5 L and about 2,000 L, between about 5 L and about 1,500 L,between about 5 L and about 1,000 L, or between about 5 L and about 500L). Culturing cells can be performed, e.g., using a fed-batch bioreactoror a perfusion bioreactor. Non-limiting examples and different aspectsof culturing cells (e.g., culturing mammalian cells) are described belowand can be used in any combination.

Cells

The cells that are cultured in some of the processes described hereincan be bacteria (e.g., gram negative bacteria), yeast (e.g.,Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha,Kluyveromyces lactis, Schizosaccharomyces pombe, Yarrowia lipolytica, orArxula adeninivorans), or mammalian cells. The mammalian cell can be acell that grows in suspension or an adherent cell. Non-limiting examplesof mammalian cells that can be cultured in any of the processesdescribed herein include: Chinese hamster ovary (CHO) cells (e.g., CHODG44 cells or CHO-Kls cells), Sp2.0, myeloma cells (e.g., NS/0),B-cells, hybridoma cells, T-cells, human embryonic kidney (HEK) cells(e.g, HEK 293E and HEK 293F), African green monkey kidney epithelialcells (Vero) cells, and Madin-Darby Canine (Cocker Spaniel) kidneyepithelial cells (MDCK) cells. In some examples where an adherent cellis cultured, the culture can also contain a plurality of microcarriers(e.g., microcarriers that contain one or more pores). Additionalmammalian cells that can be cultured in any of the processes describedherein are known in the art.

The mammalian cell can contain a recombinant nucleic acid (e.g., anucleic acid stably integrated in the mammalian cell's genome) thatencodes a recombinant therapeutic protein. Non-limiting examples ofrecombinant nucleic acids that encode exemplary recombinant therapeuticproteins are described below, as are recombinant therapeutic proteinsthat can be produced using the methods described herein. In someinstances, the mammalian cell that is cultured in a bioreactor (e.g.,any of the bioreactors described herein) was derived from a largerculture.

A nucleic acid encoding a recombinant therapeutic protein can beintroduced into a mammalian cell using a wide variety of methods knownin molecular biology and molecular genetics. Non-limiting examplesinclude transfection (e.g., lipofection), transduction (e.g.,lentivirus, adenovirus, or retrovirus infection), and electroporation.In some instances, the nucleic acid that encodes a recombinanttherapeutic protein is not stably integrated into a chromosome of themammalian cell (transient transfection), while in others the nucleicacid is integrated. Alternatively or in addition, the nucleic acidencoding a recombinant therapeutic protein can be present in a plasmidand/or in a mammalian artificial chromosome (e.g., a human artificialchromosome). Alternatively or in addition, the nucleic acid can beintroduced into the cell using a viral vector (e.g., a lentivirus,retrovirus, or adenovirus vector). The nucleic acid can be operablylinked to a promoter sequence (e.g., a strong promoter, such as aβ-actin promoter and CMV promoter, or an inducible promoter). A vectorcontaining the nucleic acid can, if desired, also contain a selectablemarker (e.g., a gene that confers hygromycin, puromycin, or neomycinresistance to the mammalian cell).

In some instances, the recombinant therapeutic protein is a secretedprotein and is released by the mammalian cell into the extracellularmedium (e.g., the first and/or second liquid culture medium). Forexample, a nucleic acid sequence encoding a soluble recombinanttherapeutic protein can contain a sequence that encodes a secretionsignal peptide at the N- or C-terminus of the recombinant therapeuticprotein, which is cleaved by an enzyme present in the mammalian cell,and subsequently released into the extracellular medium (e.g., the firstand/or second liquid culture medium).

Culture Media

Liquid culture media are known in the art. The liquid culture media(e.g., a first and/or second tissue culture medium) can be supplementedwith a mammalian serum (e.g., fetal calf serum and bovine serum), and/ora growth hormone or growth factor (e.g., insulin, transferrin, andepidermal growth factor). Alternatively or in addition, the liquidculture media (e.g., a first and/or second liquid culture medium) can bea chemically-defined liquid culture medium, an animal-derived componentfree liquid culture medium, a serum-free liquid culture medium, or aserum-containing liquid culture medium. Non-limiting examples ofchemically-defined liquid culture media, animal-derived component freeliquid culture media, serum-free liquid culture media, andserum-containing liquid culture media are commercially available.

A liquid culture medium typically contains an energy source (e.g., acarbohydrate, such as glucose), essential amino acids (e.g., the basicset of twenty amino acids plus cysteine), vitamins and/or other organiccompounds required at low concentrations, free fatty acids, and/or traceelements. The liquid culture media (e.g., a first and/or second liquidculture medium) can, if desired, be supplemented with, e.g., a mammalianhormone or growth factor (e.g., insulin, transferrin, or epidermalgrowth factor), salts and buffers (e.g., calcium, magnesium, andphosphate salts), nucleosides and bases (e.g., adenosine, thymidine, andhypoxanthine), protein and tissue hydrolysates, and/or any combinationof these additives.

A wide variety of different liquid culture media that can be used toculture cells (e.g., mammalian cells) in any of the methods describedherein are known in the art. Medium components that also may be usefulin the present processes include, but are not limited to,chemically-defined (CD) hydrolysates, e.g., CD peptone, CD polypeptides(two or more amino acids), and CD growth factors. Additional examples ofliquid tissue culture medium and medium components are known in the art.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium described herein can be thesame type of media or different media.

Additional Features of Exemplary Bioreactors

The interior surface of any of the bioreactors described herein may haveat least one coating (e.g., at least one coating of gelatin, collagen,poly-L-ornithine, polystyrene, and laminin), and as is known in the art,one or more ports for the sparging of O₂, CO₂, and N₂ into the liquidculture medium, and a stir mechanism for agitating the liquid culturemedium. The bioreactor can incubate the cell culture in a controlledhumidified atmosphere (e.g., at a humidity of greater than 20%, 30%,40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or a humidity of 100%).The bioreactor can also be equipped with a mechanical device that iscapable of removing a volume of liquid culture medium from thebioreactor and optionally, a filter within the mechanical device thatremoves the cells from the liquid culture medium during the process oftransfer of the liquid culture medium out of the bioreactor (e.g., anATF system).

Temperature

The step of culturing of mammalian cells can be performed at atemperature of about 31° C. to about 40° C. Skilled practitioners willappreciate that the temperature can be changed at specific time point(s)in during the culturing step, e.g., on an hourly or daily basis. Forexample, the temperature can be changed or shifted (e.g., increased ordecreased) at about one day, two days, three days, four days, five days,six days, seven days, eight days, nine days, ten days, eleven days,twelve days, fourteen days, fifteen days, sixteen days, seventeen days,eighteen days, nineteen days, or about twenty days or more after theinitial seeding of the bioreactor with the cell (e.g., mammalian cell).For example, the temperature can be shifted upwards (e.g., a change ofup to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5,2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to or about 20degrees C.). For example, the temperature can be shifted downwards(e.g., a change of up to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, orup to or about 20° C.).

CO₂

The culturing step described herein can further include exposing theliquid culture medium in the bioreactor to an atmosphere containing atmost or about 15% CO₂ (e.g., at most or about 14% CO₂, 12% CO₂, 10% CO₂,8% CO₂, 6% CO₂, 5% CO₂, 4% CO₂, 3% CO₂, 2% CO₂, or at most or about 1%CO₂).

Perfusion Bioreactor

The culturing step described herein can be performed using a perfusionbioreactor. Culturing a cell (e.g., a mammalian cell) in a perfusionbioreactor includes the removal from the bioreactor of a first volume ofa first liquid culture medium (e.g., containing any concentration ofmammalian cells, e.g., a first volume of a first liquid culture mediumthat is substantially free of cells), and adding to the first liquidculture medium a second volume of a second liquid culture medium.Removal and adding can be performed simultaneously or sequentially, or acombination of the two. Further, removal and adding can be performedcontinuously (e.g., at a rate that removes and replaces a volume ofbetween 0.1% to 800% (e.g., between 1% and 700%, between 1% and 600%,between 1% and 500%, between 1% and 400%, between 1% and 350%, between1% and 300%, between 1% and 250%, between 1% and 100%, between 100% and200%, between 5% and 150%, between 10% and 50%, between 15% and 40%,between 8% and 80%, and between 4% and 30%) of the volume of thebioreactor or the first liquid culture medium volume over any given timeperiod (e.g., over a 24-hour period, over an incremental time period ofabout 1 hour to about 24 hours, or over an incremental time period ofgreater than 24 hours)) or periodically (e.g., once every third day,once every other day, once a day, twice a day, three times a day, fourtimes a day, or five times a day), or any combination thereof. Whereperformed periodically, the volume that is removed or replaced (e.g.,within about a 24-hour period, within an incremental time period ofabout 1 hour to about 24 hours, or within an incremental time period ofgreater than 24 hours) can be, e.g., between 0.1% to 800% (e.g., between1% and 700%, between 1% and 600%, between 1% and 500%, between 1% and400%, between 1% and 300%, between 1% and 200%, between 1% and 100%,between 100% and 200%, between 5% and 150%, between 10% and 50%, between15% and 40%, between 8% and 80%, and between 4% and 30%) of the volumeof the bioreactor or the first liquid culture medium volume. The firstvolume of the first liquid culture medium removed and the second volumeof the second liquid culture medium added can in some instances be heldapproximately the same over each 24-hour period (or, alternatively, anincremental time period of about 1 hour to about 24 hours or anincremental time period of greater than 24 hours) over the entire orpart of the culturing period. As is known in the art, the rate at whichthe first volume of the first liquid culture medium is removed(volume/unit of time) and the rate at which the second volume of thesecond liquid culture medium is added (volume/unit of time) can bevaried. The rate at which the first volume of the first liquid culturemedium is removed (volume/unit of time) and the rate at which the secondvolume of the second liquid culture medium is added (volume/unit oftime) can be about the same or can be different.

Alternatively, the volume removed and added can change (e.g., graduallyincrease) over each 24-hour period (or alternatively, an incrementaltime period of between 1 hour and about 24 hours or an incremental timeperiod of greater than 24 hours) during the culturing period. Forexample the volume of the first liquid culture medium removed and thevolume of the second liquid culture medium added within each 24-hourperiod (or alternatively, an incremental time period of between about 1hour and above 24 hours or an incremental time period of greater than 24hours) over the culturing period can be increased (e.g., gradually orthrough staggered increments) over the culturing period from a volumethat is between 0.5% to about 20% of the bioreactor volume or the firstliquid culture medium volume to about 25% to about 150% of thebioreactor volume or the first liquid culture medium volume.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium can be the same type ofmedia. In other instances, the first liquid culture medium and thesecond liquid culture medium can be different.

The first volume of the first liquid culture medium can be removed,e.g., by a mechanical system that can remove the first volume of thefirst liquid culture medium from the bioreactor (e.g., the first volumeof the first liquid culture medium that is substantially free of cellsfrom the bioreactor). Alternatively or in addition, the first volume ofthe first liquid culture medium can be removed by seeping or gravityflow of the first volume of the first liquid culture medium through asterile membrane with a molecular weight cut-off that excludes the cell(e.g., mammalian cell).

The second volume of the second liquid culture medium can be added tothe first liquid culture medium in an automated fashion, e.g., byperfusion pump.

In some instances, removing the first volume of the first liquid culturemedium (e.g., a first volume of the first liquid culture medium that issubstantially free of mammalian cells) and adding to the first liquidculture medium a second volume of the second liquid culture medium doesnot occur within at least 1 hour (e.g., within 2 hours, within 3 hours,within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8hours, within 9 hours, within 10 hours, within 12 hours, within 14hours, within 16 hours, within 18 hours, within 24 hours, within 36hours, within 48 hours, within 72 hours, within 96 hours, or after 96hours) of the seeding of the bioreactor with a mammalian cell.

Fed-Batch Bioreactor

The culturing step described herein can be performed using a fed-batchbioreactor. Culturing a cell in a fed-batch bioreactor includes, overthe majority of the culturing period, the addition (e.g., periodic orcontinuous addition) to the first liquid culture medium of a secondvolume of a second liquid culture medium. The adding of the secondliquid culture medium can be performed continuously (e.g., at a ratethat adds a volume of between 0.1% to 300% (e.g., between 1% and 250%,between 1% and 100%, between 100% and 200%, between 5% and 150%, between10% and 50%, between 15% and 40%, between 8% and 80%, and between 4% and30%) of the volume of the bioreactor or the first liquid culture mediumvolume over any given time period (e.g., over a 24-hour period, over anincremental time period of about 1 hour to about 24 hours, or over anincremental time period of greater than 24 hours)) or periodically(e.g., once every third day, once every other day, once a day, twice aday, three times a day, four times a day, or five times a day), or anycombination thereof. Where performed periodically, the volume that isadded (e.g., within about a 24-hour period, within an incremental timeperiod of about 1 hour to about 24 hours, or within an incremental timeperiod of greater than 24 hours) can be, e.g., between 0.1% to 300%(e.g., between 1% and 200%, between 1% and 100%, between 100% and 200%,between 5% and 150%, between 10% and 50%, between 15% and 40%, between8% and 80%, and between 4% and 30%) of the volume of the bioreactor orthe first liquid culture medium volume. The second volume of the secondliquid culture medium added can in some instances be held approximatelythe same over each 24-hour period (or, alternatively, an incrementaltime period of about 1 hour to about 24 hours or an incremental timeperiod of greater than 24 hours) over the entire or part of theculturing period. As is known in the art, the rate at which the secondvolume of the second liquid culture medium is added (volume/unit oftime) can be varied over the entire or part of the culturing period. Forexample, the volume of the second liquid culture medium added can change(e.g., gradually increase) over each 24-hour period (or alternatively,an incremental time period of between 1 hour and about 24 hours or anincremental time period of greater than 24 hours) during the culturingperiod. For example the volume of the second liquid culture medium addedwithin each 24-hour period (or alternatively, an incremental time periodof between about 1 hour and above 24 hours or an incremental time periodof greater than 24 hours) over the culturing period can be increased(e.g., gradually or through staggered increments) over the culturingperiod from a volume that is between 0.5% to about 20% of the bioreactorvolume or the first liquid culture medium volume to about 25% to about150% of the bioreactor volume or the first liquid culture medium volume.The rate at which the second volume of the second liquid culture mediumis added (volume/unit of time) can be about the same over the entire orpart of the culturing period.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium can be the same type ofmedia. In other instances, the first liquid culture medium and thesecond liquid culture medium can be different. The volume of the secondliquid culture medium can be added to the first liquid culture medium inan automated fashion, e.g., by perfusion pump.

In some instances, adding to the first liquid culture medium a secondvolume of the second liquid culture medium does not occur within atleast 1 hour (e.g., within 2 hours, within 3 hours, within 4 hours,within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9hours, within 10 hours, within 12 hours, within 14 hours, within 16hours, within 18 hours, within 24 hours, within 36 hours, within 48hours, within 72 hours, within 96 hours, or after 96 hours) of theseeding of the bioreactor with a mammalian cell. The cell culture mediumin fed-batch cultures is typically harvested at the end of cultureperiod and used in any of the processes described herein, however, thecell culture medium in fed-batch cultures can also be harvested at oneor more time points during the culturing period and used in any of theprocesses described herein.

Skilled practitioners will appreciate that any of the various cultureparameters (e.g., containers, volumes, rates or frequencies of replacingculture volumes, agitation frequencies, temperatures, media, and CO₂concentrations) can be used in any combination in to perform thesemethods. Further, any of the mammalian cells described herein or knownin the art can be used to produce a recombinant protein.

Additional Examples of Unit Operations

Some examples of the methods of processing a fluid including arecombinant therapeutic protein can further include performing one ormore additional unit operations (e.g., one or more of capturing arecombinant therapeutic protein, purifying a recombinant therapeuticprotein, polishing a recombinant therapeutic protein, viralinactivation, viral filtration, adjusting the ionic concentration and/orpH of a fluid containing the recombinant therapeutic protein, filtrationof a fluid containing the recombinant therapeutic protein, precipitationand flocculation, aqueous two-phase extraction, lyophilization, andcrystallization). Some examples of the integrated and continuousprocesses for manufacturing a therapeutic protein drug substance canfurther include one or more additional unit operations, e.g., one ormore of precipitation and flocculation, aqueous two-phase extraction,lyophilization, and crystallization.

Precipitation and Flocculation

In some examples, the processes and methods described herein furtherinclude performing the unit operation(s) of precipitation and/orflocculation. Methods for performing precipitation are described in,e.g., McNerney et al., PDADMAC Flocculation of Chinese Hamster OvaryCells: Enabling a Centrifuge-Less Harvest Process for MonoclonalAntibodies, in MAbs, vol. 2, Taylor & Francis, pp. 413-427, 2015, Roushet al., Biotechnol. Prog. 24(3):488-495, 2008; Liu et al., Recovery andPurification Process Development for Monoclonal Antibody Production, inMAbs, vol. 5, Taylor & Francis, pp. 480-499, 2010; Tscheliessnig et al.,J. Biotechnol. 188:17-28, 2014; Sommer et al., Process Biochem. 49(11):2001-2009, 2014; Brodsky et al., Biotechnol. Bioeng. 109(10):2589-2598, 2012; Lydersen et al., Ann. NY Acad. Sci.745(1):222-231, 1994; and Ito et al., J. Chromatogr. B 878(2):154-164,2010. Precipitation methods can be achieved by altering factors like pHand/or conductivity (salting-in or salting-out) of solution, or throughaddition of precipitants, e.g., ethanol, ammonium sulfate, polyethyleneglycol (PEG), caprylic acid, and divalent ions. Methods for performingflocculation are described in, e.g., Riske et al., J. Biotechnol.128(4):813-823, 2007; Kang et al., Biotechnol. Bioeng.110(11):2928-2937, 2013; Singh et al., Biotechnol. Bioeng.113(4):698-716, 2015; Gagnon et al., J. Chromatogr. A 1221:57-70, 2012;Buchacher et al., Biotechnol. J. 1(2):148-163, 2006; Watt et al., VoxSang. 18(1):42-61, 1970; Bell et al., The formation of proteinprecipitates and their centrifugal recovery, in Downstream Processing,Springer, pp. 1-72, 1983; Chang et al., Biotechnol. Bioeng.31(8):841-846, 1988; Ito et al., J. Chromatogr. B 878(2):154-164, 2010;Hammerschmidt et al., Biotechnol. J. 10(8):1196-1205, 2015;Hammerschmidt et al., Process Biochem. 51(2):325-332, 2016;Hammerschmidt et al., Biotechnol. J. 9(6):766-775, 2014; and Warikoo etal., Biotechnol. J. 10(8): 1101-1102, 2015). Flocculation relies onflocculating agents, e.g., polyelectrolytes, which cause adhesion ofdispersed particulates into larger-size clusters, resulting in anincrease in the average particle size distribution. The floccules canthen be collected by filtration, sedimentation, or centrifugation.

Continuous Aqueous Two-Phase Extraction

In some examples, the processes and methods described herein furtherinclude performing the unit operation(s) of aqueous two-phaseextraction, e.g., as described in Azevedo et al., Sep. Purif Technol.65(1):14-21, 2009; Gomes et al., Sep. Purif Technol. 65(1):22-30, 2009;Kumar et al., Biotechnol. Bioeng. 75(5):570-580, 2001; Mashayekhi etal., Biotechnol. Bioeng. 102(6):1613-1623, 2009; Rosa et al., J.Chromatogr. A 1217(16):2296-2305, 2010; Haraguchi et al., Fluid PhaseEquilibria 215(1):1-15, 2004; Hart et al., Nat. Biotechnol. 12(11):1113-1117, 1994; and Azevedo et al., Trends Biotechnol. 27(4):240-247,2009b).

Aqueous two-phase extraction relies on formation of two immiscibleaqueous phases such that the recombinant therapeutic protein partitionsinto one phase while impurities partition into the other. Organicsolvent mixtures are not suitable as proteins can denature in organicphase. Hence, aqueous two-phase extraction for recombinant therapeuticproteins is usually carried out with either polymer-polymer or apolymer-salt dissolved in water above critical concentration (Azevedo etal., Trends Biotechnol. 27(4):240-247, 2009). Polymers, such as PEG,polyvinyl alcohol, dextran, and starch, and buffering salts, such asphosphates, sulfates, and citrates, have been used to create the twophases. To improve the specificity of the recombinant therapeuticprotein to a particular phase, functionalization of the phase-formingcomponent is also possible (Ruiz-Ruiz et al., J. Chromatogr. A1244:1-13, 2012).

The mechanism of phase extraction in aqueous two phase systems and inconventional organic-aqueous systems is similar; hence most of thestandard extraction equipment used in chemical industry can be appliedfor aqueous two-phase extraction (Kula and Selber, Encyclopedia ofBioprocess Technology, 2002, and Vazquez-Villegas et al., Sep. PurifTechnol. 141:263-268, 2015). Aqueous two-phase extraction can beperformed using appropriate column contactors, spray columns, rotatingdisk contactors, or mixer-settler units (Rosa et al., J. Chromatogr. B880:148-156, 2012; Muendges et al., Biotechnol. Prog. 31 (4):925-936,2015; and Espitia-Saloma et al., Food Bioprod. Process 92(2): 101-112,2014). Use of multi-stage aqueous two-phase extraction devices such asmulti-plate column contactors and mixer-settler configurations canovercome the limitations of single stage extraction through improvedrecovery and/or purity (Espitia-Saloma et al., Biotechnol. J.11(5):708-716, 2016, and Rosa et al., J. Biotechnol. 139(4):306-313,2009). Multi-stage aqueous two-phase extraction has been successfullyevaluated for the purification of mAbs (Muendges et al., Biotechnol.Prog. 31(4):925-936, 2015; Rosa et al., Biotechnol. J. 8(3):352-362,2013; Rosa et al., J. Biotechnol. 139(4):306-313, 2009; Eggersgluess etal., Chemical Engineering & Technology 37(4):675-682, 2014). ContinuousATPE devices provide a promising non-chromatographic alternative for usein downstream processing of monoclonal antibodies; allowingsimultaneously clarification, concentration and partial purification inone unit. One of the concerns on the application of ATPE at industrialscale is related to the high consumption of polymers and salt needed inthe process and, consequently, to their impact on water treatment(Espitia-Saloma et al., Biotechnol. J. 11(5):708-716, 2016).

Lyophilization

In some examples, the processes and methods described herein furtherinclude performing the unit operation of lyophilization (e.g.,continuous lyophilization). Methods for performing continuouslyophilization is described in De Meyer et al., Int. J. Pharm.496(1):75-85, 2015; Weisselberg, U.S. Pat. No. 8,528,225, 2013; and Reyet al., Glimpses into the Realm of Freeze-Drying: Classical Issues andNew Ventures, in Freeze Drying/Lyophilization of Pharmaceutical andBiological Products, Informa Healthcare, London, UK, pp. 1-28, 2010.

Crystallization

In some examples, the processes and methods described herein furtherinclude performing the unit operation of crystallization (e.g.,continuous crystallization). Methods for performing crystallization aredescribed in, e.g., Peters et al., Protein Expression Purif 39(1):43-53,2005; Schmidt et al., Eng. Life Sci. 5(3):273-276, 2005; Hekmat,Bioprocess Biosystems Eng. 38(7): 1209-1231, 2015; Basu et al., ExpertOpin. Biol. Ther. 4(3):301-317, 2004; Hekmat, Bioprocess Biosystems Eng.38(7): 1209-1231, 2015; Power et al., Chem. Eng. Sci. 133:125-139, 2015;Lawton et al., Organic Process Research Development 13(6):1357-1363,2009; Wong et al., Crystal Growth Design 12(11):5701-5707, 2012; Li etal., Organic Process Research Development 20(2):510-516, 2015;Poechlauer et al., Organic Process Research Development16(10):1586-1590, 2012; Mascia et al., Angewandte Chemie InternationalEdition 52(47): 12359-12363, 2013; Furuta et al., Chem. Eng. Process102:210-218, 2016; Power et al., Chem. Eng. Sci. 133:125-139, 2015; andNeugebauer et al., Crystal Growth Design 15(3):1089-1095, 2015).Continuous crystallization can be performed using mixed-suspensionmixed-product removal reactors (MSMPR), plug flow reactors (PFRs), andcontinuous oscillatory baffled crystallizers (COBCs) (see, e.g., Su etal., Chemical Engineering Processing: Process Intensification 89:41-53,2015).

Exemplary Types of Chromatography

Exemplary types of chromatography that can be used in the presentmethods and processes (e.g., in the unit operations performed in themethods and processes described herein) include continuous cross-currentchromatography (e.g., continuous annular chromatography and continuousradial flow chromatography), continuous counter-current chromatography(e.g., simulated moving bed chromatography, periodic counter-currentchromatography, continuous counter-current tangential chromatography,and multicolumn counter-current solvent gradient purification), andflow-through chromatography (e.g., expanded bed chromatography).

In cross-current chromatography, the resin bed moves perpendicular tothe direction of the liquid flow within the bed. In continuouscross-current systems, a steady-state separation occurs in the axial andcircumferential direction, which is in contrast to conventional batchsystems where separation occurs only in axial direction (Bridges andBarker, Continuous Cross-Current Chromatographic Refiners, in Ganetsoset al. (Eds.), Preparative and Production Scale Chromatography, Vol. 61,Marcel Dekker, Inc., New York, N.Y., pp. 113-126, 1992). Non-limitingexamples of cross-current chromatography are continuous annularchromatography and continuous radial flow chromatography.

Continuous annular chromatography allows for the continuous separationof multicomponent mixtures using a rotating annular bed ofchromatography matrix. Methods for performing continuous annularchromatography are described in, e.g., Martin, Discussions of theFaraday Society 7:332-336, 1949; Giddings, Anal. Chem. 34 (3):314-319,1962; Fox et al., J. Chromatogr. A 43:48-54, 1969; Fox, J. Chromatogr. A43:55-60, 1969; and Nicholas and Fox, J. Chromatogr. A 43:61-65, 1969;Giovannini and Freitag, Biotechnol. Bioeng. 73 (6):522-529, 2001;Bloomingburg and Carta, Chem. Eng. J. 55(1-2):B19-B27, 1994; Takahashiand Goto, Separation Sci. Technol. 26(1):1-13, 1991; and Vogel et al.,Biotechnol. Bioeng. 80(5):559-568, 2002. Continuous annularchromatography typically uses a bed of conventional adsorbent which ispacked between two concentric cylinders that forms the annulus. Theannular bed slowly rotates about its vertical axis. The feed mixture tobe separated is introduced continuously through a stationary nozzle ontop of the moving adsorbent bed. In the simplest case of isocraticchromatography, the rotating annulus is flooded with elution bufferwhich is flowing in the direction of feed (cross-current flow). As timeprogresses, different components of the feed separate in axial directionand develop helical bands from the fixed inlet point to the bottom ofthe annular bed. Several fixed outlets are kept at the bottom of the bedwhere the separated components are continuously recovered. The angulardisplacement of each component band from the feed point, i.e. slope ofthe helical bands is dependent upon the flow rate, rotational speed ofthe annulus, and the component affinity for the stationary phase.

Continuous radial flow chromatography operates similarly to continuousannular chromatography except feed and all necessary buffers flow in theradial direction (inwards from periphery or outwards from center inhorizontal direction) instead of axial direction (along the axis).Effective bed height of a radial flow column is the distance between theinner and outer cylinders while cross sectional area is associated withthe surface area of the tube. Short bed depth and large columncross-sectional area enables separations at high flow rates whilemaintaining low pressure drops (Besselink et al., J. Chromatogr. A1271(1):105-114, 2013). Additional aspects of continuous radial flowchromatography are described in, e.g., Cabanne et al., J. Chromatogr. B845 (2):191-199, 2007; Kinna et al., Biotechnol. Bioeng. 113(1):130-140, 2016; Weaver et al., BioPharm 3(7):25-28, 1990; Sun et al.,Biomed. Chromatogr. 14(7):478-482, 2000; Tharakan and Belizaire, J.Chromatogr. A 702(1):191-196, 1995; Gu et al., Chem. Eng. Sci.46(5):1279-1288, 1991; Huang et al., Chem. Eng. J. 38(3):179-186, 1988;Kinna et al., Biotechnol. Bioeng. 113(1):130-140, 2016; and Lay et al.,Food Bioprod. Process. 84(1):78-83, 2006.

Non-limiting examples of continuous counter-current chromatographyinclude simulating moving bed chromatography, periodic counter-currentchromatography, continuous counter-current tangential chromatography,and multicolumn counter-current solvent gradient purification.

Counter-current chromatography allows stationary phase to be used moreefficiently improving productivity and decreasing solvent/bufferconsumption as compared to batch chromatography. The liquid-solidcounter-current movement could be obtained by rotating the solid phaseadsorbent (chromatography resin) in the opposite direction to the liquidphase flow. This can be achieved by physically rotating the solidadsorbent/columns in the opposite direction to the liquid flow (truemoving bed) or by simulating the continuous movement of solid phaseusing an elaborate column and valve movement in a periodic orsemi-continuous manner (simulated moving bed).

Simulating moving bed chromatography utilizes a flow scheme that takesadvantages of continuous and countercurrent movement of liquid and solidphase without actual movement of the adsorbent. Instead of a largepacked bed used in traditional batch chromatography; SMB chromatographysystems are characterized by the connection of multiple smaller packedbeds. The simulated counter-current flow is carried out through acomplex valve system located between the columns, such that inlet andoutlet positions are periodically switched from column to column in thedirection of liquid flow. Methods of using simulated moving bedchromatography are described in, e.g., Jungbauer et al., TrendsBiotechnol. 31(8):479-492, 2013; Low et al., J. Chromatogr. B848(1):48-63, 2007; Xie et al., Biotechnol. Bioprocess Eng.6(6):363-375, 2001; Imamoglu, Simulated Moving Bed Chromatography (SMB)for Application in Bioseparation, in Modern Advances in Chromatography,Springer, pp. 211-231, 2002; Mun et al., Ind. Eng. Chem. Res. 42(9):1977-1993, 2003; Rajendran et al., J. Chromatogr. A 1216(4):709-738, 2009; Gottschlich and Kashe, J. Chromatogr. A765(2):201-206, 1997; Kepler et al., J. Chromatogr. A 1176 (1):69-78,2007; Krober et al., J. Chromatogr. A 1307:99-110, 2013; Rodrigues,Simulated Moving Bed Technology: Principles, Design, and ProcessApplications, Butterworth-Heinemann, 2015; and Aniceto et al.,Separation Purification Reviews 44(1):41-73, 2015.

Another example of counter-current chromatography is periodiccounter-current chromatography. Exemplary methods for performingcounter-current chromatography are described herein. Additional methodsfor performing counter-current chromatography are described in, e.g.,Mahajan et al., J. Chromatogr. A 1227:154-162, 2012; Warikoo et al.,Biotechnol. Bioeng. 109(12):3018-3029, 2012; and Godawat et al.,Biotechnol. J. 7(12):1496-1508, 2012.

Continuous counter-current tangential chromatography is a type ofcounter-current chromatography whereby, instead of packing the resinparticles in a fixed bed column, the resin (in the form of a slurry)flows through a series of static mixers and hollow fiber membranemodules. The microporous hollow fiber membranes retain the large resinparticles while letting all dissolved species (protein and buffercomponents) pass through the membrane and into the permeate. The buffersused in the binding, washing, elution, stripping, and equilibrationsteps flow countercurrent to the resin slurry in a multi-stageconfiguration. Methods of performing continuous counter-currenttangential chromatography are described in, e.g., Dutta et al.,Biotechnol. Prog. 32(2):430-439, 2016; Shinkazh et al., U.S. Pat. No.7,988,859, 2011; Napadensky et al., Separation Science Technology 48(9):1289-1297, 2013; and Dutta et al., J. Biotechnol. 213:54-64, 2015.

Multicolumn counter-current solvent gradient purification is amulticolumn chromatographic process that is capable of using lineargradients for high-resolution separation of three component fractions.Methods for performing multicolumn counter-current solvent gradientpurification are described in Aumann and Morbidelli, Biotechnol. Bioeng.98 (5):1043-1055, 2007; Muiller-Spath et al., Biotechnol. Bioeng.100(6):1166-1177, 2008; and Aumann and Morbidelli, Biotechnol. Bioeng.99 (3):728-733, 2008.

An example of flow-through chromatography is expanded bedchromatography. Expanded-bed chromatography (EBC) utilizes a fluidizedchromatographic adsorbent bed, which allows desired proteins to bepurified directly from crude (e.g., particulate containing) feedstock(Chase, Trends Biotechnol. 12(8):296-303, 1994; Thommes, Fluidized BedAbsorption as a Primary Recovery Step in Protein Purification, in NewEnzymes for Organic Synthesis, Springer, pp. 185-230, 1997; Anspach etal., J. Chromatogr. A 865 (1):129-144, 1999; and Gagnon, J. Chromatogr.A 1221:57-70, 2012). The primary difference in setup of expanded bedchromatography compared to traditional chromatography columns is thatthe top flow adapter is appropriately positioned such that the bed isallowed to expand upward in the direction of liquid flow. Methods forperforming expanded bed chromatography are described in, e.g., Kinna etal., Biotechnol. Bioeng. 113(1):130-140, 2016; Farid, J. Chromatogr. B848(1):8-18, 2007; Chhatre et al., Bioprocess Biosystems Eng.30(1):1-11, 2007; Lin et al., J. Chromatogr. A 1304:78-84, 2013; Zhao etal., Chinese J. Chem. Eng. 17(4):678-687, 2009; Smith et al., J.Chromatogr. A 968 (1):121-128, 2002; Feuser et al., Process Biochem.34(2):159-165, 1999; Ozyurt et al., J. Chromatogr. A 944(1):203-210,2002; Anspach et al., J. Chromatogr. A 865(1):129-144, 1999; Owen andChase, J. Chromatogr. A 757 (1):41-49, 1997; and Owen and Chase, Chem.Eng. Sci. 54(17):3765-3781, 1999.

Exemplary Advantages

The processes described herein can result in a substantial increase inthe volumetric productivity of the recombinant therapeutic proteinpresent in the therapeutic protein drug substance. For example, theprocesses described herein can result at least a 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, and 10-fold increase in thevolumetric productivity of the recombinant therapeutic protein presentin the therapeutic protein drug substance. The biological activity of arecombinant therapeutic protein can be assessed using a variety ofmethods known in the art, and will depend on the activity of thespecific recombinant therapeutic protein. For example, the biologicalactivity of a recombinant therapeutic protein that is an immunoglobulin(e.g., an antibody or an antibody fragment) can be determined bymeasuring the affinity of the recombinant therapeutic antibody to bindto its specific epitope (e.g., using Biocore or competitiveenzyme-linked immunosorbent assays). The recombinant therapeutic proteinmay be an enzyme (e.g., a recombinant galactosidase, e.g., a recombinantalpha-galactosidase) and the biological activity may be determined bymeasuring the recombinant therapeutic enzyme's activity (e.g.,determining the catalytic rate constant of the recombinant therapeuticenzyme by measuring a decrease in the concentration of a detectablesubstrate or an increase in the concentration of a detectable product(e.g., using spectrophotometry or light emission). For example, thebiological activity of a recombinant therapeutic galactosidase can bedetected by measuring a decrease in the level of globotriasylceramide(GL-3) or galabiosylceramide, or an increase in the level of ceramidedihexoside or galactose.

The processes described herein can result in an increased percentage ofrecovery of the recombinant therapeutic protein (e.g., increasedpercentage of yield of the recombinant therapeutic protein present inthe liquid culture medium in the therapeutic protein drug substance).For example, the present processes can result in a percentage yield ofrecombinant therapeutic protein of greater than about 70%, greater thanabout 80%, greater than about 82%, greater than about 84%, greater thanabout 86%, greater than about 88%, greater than about 90%, greater thanabout 92%, greater than about 94%, greater than about 96%, or greaterthan about 98%. The present processes can result in a percentage yieldof between about 80% to about 90%, between about 82% to about 90%,between about 84% to about 90%, between about 84% to about 88%, betweenabout 84% to about 94%, between about 82% to about 92%, or between about85% to about 95%.

The concentration of recombinant therapeutic protein present in thetherapeutic protein drug substance can be greater than about 1.0 mg/mL,greater than about 1.5 mg/mL, greater than about 2.0 mg/mL, greater thanabout 2.5 mg/mL, greater than about 3.0 mg/mL, greater than about 3.5mg/mL, greater than about 4.0 mg/mL, greater than about 4.5 mg/mL,greater than about 5.0 mg/mL, greater than about 5.5 mg/mL, greater thanabout 6.0 mg/mL, greater than about 6.5 mg/mL, greater than about 7.0mg/mL, greater than about 7.5 mg/mL, greater than about 8.0 mg/mL,greater than about 8.5 mg/mL, greater than about 9.0 mg/mL, greater thanabout 10.0 mg/mL, greater than about 12.5 mg/mL, or greater than about15.0 mg/mL.

The processes described herein can result in a net yield of recombinanttherapeutic protein in the therapeutic protein drug substance of atleast about 5 g/day, at least about 6 g/day, at least about 7 g/day, atleast about 8 g/day, at least about 9 g/day, at least about 10 g/day, atleast about 11 g/day, at least about 12 g/day, at least about 13 g/day,at least about 14 g/day, at least about 15 g/day, at least about 16g/day, at least about 17 g/day, at least about 18 g/day, at least about19 g/day, at least about 20 g/day, at least about 25 g/day, at leastabout 30 g/day, at least about 35 g/day, or at least about 40 g/day overa continuous period of at least about 5 days, at least about 10 days, atleast about 15 days, at least about 20 days, at least about 25 days, atleast about 30 days, at least about 35 days, at least about 40 days, atleast about 45 days, at least about 50 days, at least about 55 days, atleast about 60 days, at least about 65 days, at least about 70 days, atleast about 75 days, at least about 80 days, at least about 85 days, atleast about 90 days, at least about 95 days, at least about 100 days, atleast about 110 days, at least about 120 days, at least about 130 days,at least about 140 days, at least about 150 days, at least about 160days, at least about 170 days, at least about 180 days, at least about190 days, at least about 200 days, at least about 210 days, at leastabout 220 days, at least about 230 days, at least about 240 days, atleast about 250 days, at least about 260 days, at least about 270 days,at least about 280 days, at least about 290 days, at least about 300days, at least about 310 days, at least about 320 days, at least about330 days, at least about 340 days, at least about 350 days, or at leastabout 365 days.

The processes provided herein can result in a significantly improvedspecific productivity rate. For example, the specific productivity rateachieved in the recombinant protein drug substance is at least 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold,170-fold, 180-fold, 190-fold, or 200-fold greater than the specificproductivity rate achieved using a different process (e.g., a batchpurification process or a process that is not integrated and/orcontinuous). The productivity in the recombinant protein drug substanceachieved by the present processes can be at least 10,000 units/L, atleast 15,000 units/L, at least about 20,000 units/L, at least about25,000 units/L, at least about 30,000 units/L, at least about 35,000units/L, or at least about 40,000 units/L (in the first and/or secondliquid culture medium). The productivity in the recombinant protein drugsubstance achieved by the present methods can be at least 1 g/L, atleast 1.5 g/L, at least 2.0 g/L, at least 2.5 g/L, at least 3.0 g/L, atleast 4.0 g/L, at least 4.5 g/L, or at least 5.0 g/L.

The processes described herein also provide for time-efficientproduction of a therapeutic drug substance from a liquid culture medium.For example, the elapsed time between feeding a fluid (e.g., a liquidculture medium) containing a therapeutic protein into the first MCCS andeluting a therapeutic protein drug substance (containing the therapeuticprotein) from the outlet of the second MCCS is, e.g., between about 4hours and about 48 hours, inclusive, e.g., between about 4 hours andabout 40 hours, between about 4 hours and about 35 hours, between about4 hours and about 30 hours, between about 4 hours and about 28 hours,between about 4 hours and about 26 hours, between about 4 hours andabout 24 hours, between about 4 hours and about 22 hours, between about4 hours and about 20 hours, between about 4 hours and about 18 hours,between about 4 hours and about 16 hours, between about 4 hours andabout 14 hours, between about 4 hours and about 12 hours, between about6 hours and about 12 hours, between about 8 hours and about 12 hours,between about 6 hours and about 20 hours, between about 6 hours andabout 18 hours, between about 6 hours and about 14 hours, between about8 hours and about 16 hours, between about 8 hours and about 14 hours,between about 8 hours and about 12 hours, between about 10 hours and 20hours, between about 10 hours and 18 hours, between about 10 hours and16 hours, between about 10 hours and 14 hours, between about 12 hoursand about 14 hours, between about 10 hours and about 40 hours, betweenabout 10 hours and about 35 hours, between about 10 hours and about 30hours, between about 10 hours and about 25 hours, between about 15 hoursand about 40 hours, between about 15 hours and about 35 hours, betweenabout 15 hours and about 30 hours, between about 20 hours and about 40hours, between about 20 hours and about 35 hours, or between about 20hours and about 30 hours, inclusive. In other examples, the elapsed timebetween feeding the fluid (e.g., a liquid culture medium) containing atherapeutic protein into the first MCCS and eluting a therapeuticprotein drug substance (containing the therapeutic protein) from theoutlet of the second MCCS is, e.g., greater than about 4 hours and isless than about 40 hours, inclusive, e.g., greater than 4 hours and lessthan about 39 hours, about 38 hours, about 37 hours, about 36 hours,about 35 hours, about 34 hours, about 33 hours, about 32 hours, about 31hours, about 30 hours, about 29 hours, about 28 hours, about 27 hours,about 26 hours, about 25 hours, about 24 hours, about 23 hours, about 22hours, about 21 hours, about 20 hours, about 19 hours, about 18 hours,about 17 hours, about 16 hours, about 15 hours, about 14 hours, about 13hours, about 12 hours, about 11 hours, about 10 hours, about 9 hours,about 8 hours, about 7 hours, about 6 hours, about 5 hours, or about 4.5hours, inclusive.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Example 1. Performance of Ultrafiltration/Diafiltration Using aCircuit System

An ultrafiltration/diafiltration circuit system was created tocontinuously concentrate and buffer exchange a fluid including arecombinant bispecific antibody. The recombinant bispecific antibody wasfirst purified over a Protein A column and the eluate, at aconcentration of 24 g/L, was then used in theultrafiltration/diafiltration circuit system. The circuit systemincluded a tangential flow filtration (TFF) unit having first and secondinlets, and a conduit in fluid communication between the first andsecond inlets, including a port for flowing fluid into or out of, orboth, of the system. The fluid containing the recombinant bispecificantibody was continuously flowed through circuit system and filtrate notincluding the recombinant bispecific antibody (see “permeate” in FIG. 5)was discarded for a first period of time. A constant volume of fluid wasmaintained within the conduit during the first period of time.

Following this step, a diafiltration medium was continuously flowed intothe circuit system through the at least one port and filtrate notincluding the recombinant bispecific antibody was discarded for a secondperiod of time (FIG. 6). A constant volume of fluid was maintainedwithin the conduit during the first period of time. The mass flow ofthis circuit system in this process is depicted in FIG. 5 and FIG. 6.

The fluid including the recombinant bispecific protein in the circuitsystem at the end of the second period of time is then collected fromthe circuit system. This process resulted in a 96% recovery of therecombinant bispecific antibody. Data from experiments using anexemplary circuit system including a tangential flow filtration unit isshown in FIGS. 7-9.

Example 2. Performance of Viral Filtration Using a Circuit System

A circuit system including a tangential flow virus filtration unit wasthen created to provide for continuous removal of viruses from fluidincluding a recombinant bispecific antibody recovered from the circuitsystem including a tangential flow filtration unit described inExample 1. The pore size of the filter in the tangential flow virusfiltration unit was about 20 nm in order to provide for parvovirusremoval.

The concentrated and buffer exchanged fluid including the recombinantbispecific antibody removed from the circuit system described in Example1 was fed into a second circuit system including a tangential flow virusfiltration unit as indicated in FIG. 10. (Another example of a circuitsystem including a tangential flow virus filtration unit is shown inFIG. 11.) The fluid was continuously flowed through the second circuitsystem and the fluid including the recombinant bispecific antibody wasrecovered from the tangential flow virus filtration unit (see“virus-free” VF product in FIG. 10). Any viral particles present in thefluid flowed into the circuit system are retained within the circuitsystem and the fluid including the recombinant bispecific antibody wascollected from the tangential flow virus filtration unit. This circuitsystem was operated for about 24 hours and no flux decay or pressureincreases were observed over this period of time (see FIG. 12).

Example 3. Performance of Viral Clearance Using a Circuit SystemIncluding a TFVF Unit

The viral clearance capabilities for a circuit system including a TFVFunit were tested in several experiments using Murine Minute Virus (MMV).Filtration was performed using a test fluid included 5 mg/mL of arecombinant human antibody that was spiked with 0.5% viral material toyield a final MMV titer of approximately 6 logs, unless otherwise notedbelow. The tested TFVF units included either a 3 cm² Planova™ BioEXfilter or a 10 cm² Planova™ 20N or Planova™ BioEX filter.

An experiment was designed to test the amount of MMV virus retained bythe TFVF unit in the tested systems when the test fluid was flowed oncethrough the TFVF unit. For this experiment, the retentate was collectedin a separate pressure vessel rather than flowed back into the main feedvessel, in order to allow for a determination of the viral titer in theretentate. The system with a TFVF unit including a 10 cm² Planova™ 20Nfilter was operated at 12 psi with a flow rate varying between 1 and 2mL/min. As indicated in FIG. 13 by the linear fit to the filtrateweight, the flow rate had no impact on the filter flux rate of 45.6 LMH,meaning the flow rate can be adjusted independently to maximize membranelife. At the conclusion of the experiment, the filtrate was found torepresent 28% of volume of the test fluid, while 72% of the volume ofthe test fluid was collected in the retentate collection vessel. Theviral titer of the filtrate was below the limit of detection, while theretentate titer was higher than the titer in the starting test fluid. Amass balance for the virus indicates that when the system is operated, asignificant fraction of the virus is not captured by the filter in theTFVF unit, but flows through to concentrate in the retentate.

The viral clearance for a circuit system including a TFVF unit includinga 10 cm² Planova™ 20N filter was tested for a 22-hour period, whileoperated at 6.6 psi with a recirculation rate of 2 mL/min (FIG. 14). Thesystem was charged with an initial load of 579 mL of test fluid andfollowing operation (5.95 log titer), produced 510 mL of filtrate beforethe system was stopped to collect samples. The loading of 510 L/m² wasachieved without any flux decay, with the flux across the filterremaining a constant 23.0 LMH. The 69 mL remaining in the retentatevessel was found to be visibly cloudy at the completion of theexperiment, though no filter flux decay was found at any point duringthe run. The viral titer of the filtrate was below the limit ofdetection (<0.83 log), while the retentate titer was 6.48 log, 0.53 loghigher than the titer in the test fluid. This result represents a 3.4×concentration of the virus in the load/retentate vessel. About half ofthe total virus load was not captured by the filter, but insteadcontinued to recirculate, reducing the capture burden for the filter andpreventing flux decay.

The viral clearance for a circuit system including a TFVF unit including3 cm² or 10 cm² Planova™ BioEX filters when run for 29 hours at 20 psiwith a recirculation rate of 1 mL/min or 3 mL/min, respectively, withperiodic refeed of fresh fluid (FIGS. 15 and 16, respectively). Theexperiment performed using a TFVF unit with the 3 cm² filter wasdesigned to replicate a scenario where a large bolus of virus at hightiter is introduced to the system (e.g., through external contamination)and subsequently diluted out with virus-free material. This system wascharged with an initial bolus of ˜400 mL of test fluid spiked with 6.13logs of virus and filtered for 20 hours. The viral titer in the filtratefrom the first 20 hours of filtration was below the limit of detection(<1.09 logs). After ˜20 hours of filtration, a fresh bolus of ˜400 mLvirus-free antibody solution was added. This material was filtered foran additional 9 hours and the virus titer in the filtrate was below thelimit of detection. FIG. 15 shows the pressure and mass profiles usingthe circuit system including a TFVF including the 3 cm² Planova™ BioEXfilter. The flux remained constant at 42.8 LMH through the first 20hours but dropped to a new constant rate of 36.4 LMH after the refeed.The total throughput for the filter was 1190 L/m².

The experiment performed using the circuit system including a TFVF unitincluding the 10 cm² Planova™ BioEX filters was designed to replicate ascenario where a low titer stream of virus is continuously introducedinto the system (e.g., from a contaminated buffer). The initial virusspike material (a MMV titer of 6.13 logs) was diluted 1:30 into a freshrecombinant antibody solution to create a diluted feedstock. The viraltiter of the diluted feedstock was 4.66 logs. This diluted feedstock wasfiltered through the system for 29 hours, with fresh diluted feedstockadded periodically to maintain the working volume above a minimumthreshold. The virus titer of the filtrate was determined for the first20 hours and for the next 9 hours, and was below the limit of detection(<0.83 logs) for both samples. FIG. 16 shows the pressure and massprofiles for the experiment. The flux remained constant at 39.4 LMHthrough the first 20 hours but dropped to a new rate of 34.5 LMH afterthe feed. The total throughput for the filter was 1120 L/m².

These results indicate that operating viral filtration with a continuousrecirculation prolongs the useful life of the membrane by reducingplugging by captured virus. The ability to adjust the permeate rate (viasystem pressure) and recirculation rate (via pump speed) independentlyallow for process optimization to extend the operation of a singlefilter to multiple days.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of processing a fluid comprising a recombinant therapeuticprotein, the method comprising: (a) providing a circuit systemcomprising (i) a tangential flow filtration (TFF) unit having first andsecond inlets, and (ii) a conduit in fluid communication between thefirst and second inlets, comprising at least one port for flowing fluidinto or out of, or both, of the system, wherein the system is configuredsuch that fluid can be flowed through the conduit and the TFF unit, andfiltrate not comprising the recombinant therapeutic protein can becollected from the TFF unit; (b) continuously flowing a fluid comprisinga recombinant therapeutic protein into the circuit system through one ofthe at least one port, and discarding filtrate not comprising therecombinant therapeutic protein for a first period of time; (c)continuously flowing a diafiltration medium into the circuit systemthrough one of the at least one port, and discarding filtrate notcomprising the recombinant therapeutic protein for a second period oftime; and (d) collecting a fluid comprising the recombinant therapeuticprotein that is present in the circuit system after the first and secondtime periods through one of the at least one port.
 2. The method ofclaim 1, wherein step (b) comprises continuously flowing the fluid intothe circuit system at a rate of about 0.1 mL/minute to about 100L/minute. 3.-5. (canceled)
 6. The method of claim 1, wherein the flowingof the fluid into the circuit system in step (b) occursunidirectionally.
 7. The method of claim 1, wherein step (c) comprisingcontinuously flowing the diafiltration medium into the circuit system ata rate of about 0.1 mL/minute to about 100 L/minute. 8.-10. (canceled)11. The method of claim 1, wherein the flowing of the diafiltrationmedium into the circuit system in step (c) occurs unidirectionally.12.-13. (canceled)
 14. The method of claim 1, wherein the TFF unitcomprises one or more tangential filter(s) having a surface area ofabout 0.1 cm² to about 50 cm².
 15. (canceled)
 16. The method of claim 1,wherein circuit system further comprises a pump disposed in the conduit.17. The method of claim 16, wherein the pump is a peristaltic pump. 18.The method of claim 1, further comprising: (e) providing a secondcircuit system comprising (i) a tangential flow virus filtration (TFVF)unit having first and second inlets, and (ii) a conduit in fluidcommunication between the first and second inlets of the TFVF unit,comprising at least one port for flowing fluid into or out of, or both,of the second system, wherein the second system is configured such thatfluid can be flowed through the conduit and the TFVF unit, and filtratecomprising the recombinant therapeutic protein can be collected from theTFVF unit; and (f) flowing the collected fluid of step (d) into thesecond system using a connecting conduit in fluid communication betweena port in the system and a port in the second system, and collectingfiltrate comprising the recombinant therapeutic protein from the TFVFunit for a third period of time.
 19. The method of claim 18, whereinstep (f) comprising flowing the fluid into the second system at a rateof about 0.1 mL/minute to about 100 L/minute. 20.-22. (canceled)
 23. Themethod of claim 18, wherein the flowing of the fluid into the secondsystem in step (f) occurs unidirectionally. 24.-25. (canceled)
 26. Themethod of claim 18, wherein the TFVF unit comprises one or moretangential virus filter(s) having a surface area of about 0.1 cm² toabout 100 m². 27.-29. (canceled)
 30. The method of claim 18, whereinsecond system further comprises a pump disposed in the conduit in fluidcommunication between the first and second inlets of the TFVF unit. 31.The method of claim 30, wherein the pump disposed in the conduit influid communication between the first and second inlets of the TFVF unitis a peristaltic pump.
 32. A method of processing a fluid comprising arecombinant therapeutic protein, the method comprising: (a) providing acircuit system comprising (i) a tangential flow virus filtration (TFVF)unit having first and second inlets, and (ii) a conduit in fluidcommunication between the first and second inlets of the TFVF unit,comprising at least one port for flowing fluid into or out of, or both,of the system, wherein the system is configured such that fluid can beflowed through the conduit and the TFVF unit, and filtrate comprisingthe recombinant therapeutic protein can be collected from the TFVF unit;(b) continuously flowing a fluid including a recombinant therapeuticprotein into the circuit system through one of the at least one port,and collecting filtrate comprising the recombinant therapeutic proteinfrom the TFVF unit for a period of time.
 33. The method of claim 32,wherein step (b) comprising continuously flowing the fluid into thesystem at a rate of about 0.1 mL/minute to about 100 L/minute. 34.-36.(canceled)
 37. The method of claim 32, wherein the flowing of the fluidinto the system in step (b) occurs unidirectionally. 38.-39. (canceled)40. The method of claim 32, wherein the TFVF unit comprises one or moretangential virus filter(s) having a surface area of about 0.1 cm² toabout 100 m².
 41. (canceled)
 42. The method of claim 32, wherein systemfurther comprises a pump disposed in the conduit.
 43. The method ofclaim 42, wherein the pump disposed in the conduit is a peristalticpump.
 44. An integrated and continuous process for manufacturing atherapeutic protein drug substance, the process comprising: (a)providing a liquid culture medium comprising a recombinant therapeuticprotein that is substantially free of cells, wherein the liquid culturemedium is fed into a first multi-column chromatography system (MCCS1);(b) capturing said recombinant therapeutic protein in the liquid culturemedium using the MCCS1, wherein the eluate of the MCCS1 containing therecombinant therapeutic protein is continuously fed into a secondmulti-column chromatography system (MCCS2); (c) purifying and polishingthe recombinant therapeutic protein using the MCCS2, wherein the eluatefrom the MCCS2 is a fluid comprising the recombinant therapeuticprotein; (d) processing the fluid comprising the recombinant therapeuticprotein using a method of claim 1, wherein the process is integrated andruns continuously from said liquid culture medium to the collected fluidor filtrate comprising the recombinant therapeutic protein, wherein thecollected fluid or filtrate is the therapeutic protein drug substance.45.-72. (canceled)